Compounds and methods for use in detecting gabapentin

ABSTRACT

Compounds and methods for use in detecting gabapentin in a sample suspected of containing gabapentin are disclosed. Gabapentin derivatives are used to produce gabapentin conjugates. A gabapentin-immunogenic carrier conjugate may be used as an immunogen for the preparation of an anti-gabapentin antibody. A gabapentin-detectable label may be used in a signal producing system in gabapentin assays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/032,528, filed Feb. 15, 2008, which claims the benefit of U.S.Provisional Application No. 60/890,313, filed Feb. 16, 2007, thedisclosures of each of which applications are incorporated herein byreference.

FIELD OF THE INVENTION

The invention relates to therapeutic drug monitoring (TDM) ofgabapentin.

BACKGROUND OF THE INVENTION

Gabapentin, (1-aminomethyl)cyclohexane-1-acetic acid) is anantiepileptic drug, which has been found to have pain-relievingproperties. Gabapentin is indicated for the management of post-herpeticneuralgia in adults. It is reported as adjunctive therapy in thetreatment of partial seizures with and without secondary generalizationin patients over 12 years of age with epilepsy and in the treatment ofpartial seizures in pediatric patients age 3-12 years. Additionally, itis indicated to have anti-anxiety activity as also beneficial propertiesin treating neuro-degenerative diseases like Alzheimer's.

There can be a variable relationship between the dose of gabapentin andthe resulting serum drug concentration that provides a therapeuticeffect. This can be due to variability of intra- and inter-individualpharmacokinetics. As a result of this variability, equal doses of thesame drug in different individuals can result in dramatically differentclinical outcomes. For example, effectiveness of the same gabapentindosage can vary significantly between patients based upon individualdrug clearance and the ultimate serum drug concentration in the patient.In addition, therapeutic drug management of gabapentin can serve as anexcellent tool to ensure compliance in administering chemotherapy withthe actual prescribed dosage and achievement of the effective serumconcentration levels.

Therapeutic drug management (TDM) can provide the clinician with insighton patient variation, and allow the clinical to individualize drugdosages to the patient's needs. For example, absorption of gabapentincan be saturated as the dose is titrated, so physicians may use TDM toconfirm the plasma concentration increases with increasing doses. Thus,monitoring of levels of gabapentin in the body, and adjusting the doseas may be advised, can serve to better control therapy and limitundesirable side effects in patients.

The majority of data regarding gabapentin drug levels has been derivedusing either liquid chromatographic (Hengy & Kolle, 1985; Ratnaraj &Patsalos 1998, Chollet et al, 200, Wad & Kramer, 1998, Ifa et al. 2001)or gas chromatographic techniques (Hooper et al., 1990, Kushnir et al.,1999). While chromatographic techniques can be used to determine druglevels, such methods are impractical for commercial use due to, forexample, long sample preparation time, long assay time, high cost, andlabor-intensive procedures. Immunoassays provide simple and fastanalytical methods for measurement of drug levels.

SUMMARY OF THE INVENTION

The present disclosure provides methods and compounds for use indetecting gabapentin in a sample.

Features of the Invention

The present invention features a compound having the structure:

where R₁, R₂, or R₃ is —X—W-L-Z; where: when R₁ is —X—W-L-Z, X is NH,and R₂ is —OH, and R₃ is —H, when R₂ is —X—W-L-Z, X is NH, R₁ is —NH₂,and R₃ is —H, and when R₃ is —X—W-L-Z, X is a heteroatom or lower alkylgroup, R₁ is —NH₂, and R₂ is —OH, and W is a lower alkyl group or acarbonyl group; L is a linker or at least one bond between W and Z; andZ is H, an alkyl group, a reactive functional group capable of reactingwith a reactive partner to form a covalent bond, or a moiety ofinterest; or a salt thereof.

In some embodiments, the linker comprises 0 to 40 carbon atoms and 0-6heteroatoms. In some embodiments, W is a lower alkyl and said linker isselected from the group consisting of —(CH₂)_(n)C(O)—, —C(O)(CH₂)_(n)—,—C(O)(CH₂)_(n)NH—C(O)—, —C(O)(CH₂)_(m)NH—C(O)(CH₂)_(n)—,—(CH₂)_(n)SCH₂C(O)—, —(CH₂)_(m)SCH₂C(O)(CH₂)_(n)—,—(CH₂)_(m)C(O)NH(CH₂)_(n)—, —(CH₂)NH—C(O)—, —(CH₂)_(m)NH—C(O)(CH₂)_(n)—,—C(O)—(CH₂)_(n)—, and —(CH₂)_(n)—; wherein m, n, o, and p areindependently selected from an integer from 0 to 10.

In some embodiments, W is a carbonyl and said linker is selected fromthe group consisting of —(CH₂)_(n)C(O)—, —(CH₂)_(n)SCH₂C(O)—,—(CH₂)_(m)SCH₂C(O)(CH₂)_(n)—, —(CH₂)_(m)C(O)NH(CH₂)_(n)—,—(CH₂)NH—C(O)—, —(CH₂)_(m)NH—C(O)(CH₂)_(n)—, and —(CH₂)_(n)—; wherein m,n, o, and p are independently selected from an integer from 0 to 10. Insome embodiments, W is a methyl group.

In some embodiments, the reactive functional group is selected from thegroup consisting of halogen, OH, SH, NH₂, O-lower alkyl, epoxy, S-acyl,carboxyl, maleimidyl, haloacetamide, hydroxysuccinimidyl, succinimidyl,carbonate, anhydride and imidate. In some embodiments, the halogen is Bror I.

In some embodiments, the compound has the following structure:

wherein Z, L, and W are as defined above.

In some embodiments, Z is SH or Br. In some embodiments, Z is a reactivefunctional group, and wherein —W-L-Z is a straight or branched alkylchain. In some embodiments, Z is a reactive functional group, andwherein W is a lower alkyl group.

In some embodiments, the compound has the following structure:

wherein W, L and Z are as defined above.

In some embodiments, Z is a reactive functional group, the reactivefunctional group is selected from the group consisting of halogen, SH,NH₂, O-lower alkyl, epoxy, S-acyl, maleimidyl, haloacetamide,hydroxysuccinimidyl, succinimidyl, carbonate, anhydride and imidate.

In some embodiments, the compound has the following structure:

wherein W, L and Z are as defined above.

In some embodiments, Z is SH or Br. In some embodiments, —X—W-L-Z isselected from the group consisting of —NH—(CH₂)_(m)—SH,—NH—CO—(CH₂)_(n)—Br, and —NH—(CH₂)_(o)—S—CH₂—CO—CH₂—Br; and m, n, and oare independently selected from 2 or 3.

In some embodiments, Z is a moiety of interest. In some embodiments, themoiety of interest is an enzyme. In some embodiments, the enzyme is aglucose-6-phosphate dehydrogenase (G6PDH). In some embodiments, theG6PDH comprises at least one cysteine per subunit, and the cysteine isnot native to a naturally-occurring G6PDH. In some embodiments, theenzyme is selected from alkaline phosphatase, β-galactosidase andhorseradish peroxidase.

In some embodiments, the moiety of interest is a carrier protein. Insome embodiments, the carrier protein is bovine serum albumin (BSA). Insome embodiments, the carrier protein is keyhole limpet hemocyanin(KLH). In some embodiments, the carrier protein is selected from thegroup consisting of hemocyanins, globulins, albumins, andpolysaccharides.

In some embodiments, the compound is immunogenic.

In some embodiments, the moiety of interest is a detectable label. Insome embodiments, the detectable label is selected from a fluorophore, afluorescence quencher, a radioisotope, and metal. In some embodiments,the detectable label is selected from a polypeptide, a nucleic acid, apolysaccharide, and a lipid.

In some embodiments, the compound is immobilized on a support.

In some embodiments, the moiety of interest is bound to the compoundthrough CONH, NHCO, NHCONH, NH—(C═S)—NH, O—CO—NH, NH—O—CO, S, NH—(C═NH),N═N, or NH.

In some embodiments, the moiety of interest is selected from afluorophore, a fluorescence quencher, a radioisotope, and metal. In someembodiments, the moiety of interest is selected from a polypeptide, anucleic acid, a polysaccharide, and a lipid.

The present invention features a method for detecting the presence orabsence of gabapentin in a sample. The method generally involves:

-   -   (a) adding, to a reaction mixture, (i) a sample suspected of        containing gabapentin and (ii) an anti-gabapentin antibody        capable of forming of a complex of gabapentin that may be        present in the sample and the antibody; and    -   (b) detecting the presence or absence of the complex;    -   wherein the presence or absence of the complex is indicative of        the presence or absence of gabapentin in the sample.

In some embodiments, the method further comprises adding a gabapentinconjugate comprising a gabapentin moiety and a detectable label to saidsample, where the gabapentin conjugate is capable of binding to theanti-gabapentin antibody; and where detecting is by detecting thedetectable label.

In some embodiments, the moiety of interest is the detectable label. Insome embodiments, the detectable label comprises an enzyme and thedetecting is by assaying activity of the enzyme. In some embodiments,enzymatic activity is elevated when the complex is present. In someembodiments, the enzyme is a dehydrogenase. In some embodiments, thedehydrogenase is G6PDH.

In some embodiments, the method further comprises measuring the amountof the conjugate bound to the anti-gabapentin antibody. In someembodiments, the detecting is quantitative.

In some embodiments, the detectable label is selected from afluorophore, a fluorescence quencher, a radioisotope, and metal. In someembodiments, the detectable label is selected from a polypeptide, anucleic acid, a polysaccharide, and a lipid.

In some embodiments, the method is a homogeneous immunoassay. In someembodiments, the method is a heterogeneous immunoassay.

In some embodiments, the sample is a biological sample obtained from ahuman. In some embodiments, the biological sample is blood or ablood-derived sample.

The present invention features an antibody that specifically bindsgabapentin. In some embodiments, the antibody is selected from a Fab,Fab′, F(ab′)′2, Fv fragment, and a single-chain antibody. In someembodiments, the antibody is a monoclonal antibody.

The present invention features a kit comprising: an anti-gabapentinantibody capable of specifically binding to gabapentin; and a gabapentincalibration standard.

In some embodiments, the kit further comprises a conjugate comprising agabapentin moiety and a detectable signal. In some embodiments, thedetectable label is an enzyme, and the kit further comprises a substratefor the enzyme.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the details of the compounds and methods for use in detectinggabapentin as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures:

FIG. 1 is a schematic showing the structure of gabapentin.

FIG. 2 is a schematic showing the structures of exemplary gabapentinderivatives.

FIG. 3 is a schematic showing the structures of exemplary gabapentinconjugates comprising an exemplary protein carrier.

FIG. 4 is a schematic showing the structures of seven exemplarygabapentin conjugates comprising an exemplary enzyme.

FIG. 5 is a schematic representation of a homogeneous, competitiveimmunoassay for gabapentin using an anti-gabapentin antibody and agabapentin-G6PDH enzyme conjugate.

FIG. 6 is a calibration curve showing the change in optical densityaccording to the concentration of gabapentin in a sample.

FIGS. 7A and 7B are schematic representations of preparation of reactiveforms of KLH and an enzyme.

FIGS. 8A-K are schematic representations of synthetic schemes ofexemplary gabapentin conjugates.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Before exemplary embodiments of the invention are described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some exemplary methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited. Itis understood that the present disclosure supersedes any disclosure ofan incorporated publication to the extent there is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aconjugate” includes a plurality of such conjugates and reference to “thesample” includes reference to one or more samples and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms aredefined for purposes of the present disclosure.

A “gabapentin derivative” as used in this disclosure refers to acompound sharing a core structure with gabapentin and that can competewith gabapentin for binding to an anti-gabapentin binding partner, suchas an anti-gabapentin antibody.

Certain compounds disclosed herein in connection with embodiments of thepresent invention can exist in unsolvated forms as well as solvatedforms, including hydrated forms. In general, the solvated forms areequivalent to unsolvated forms and are encompassed within the referenceto the compounds set out in the present disclosure. Certain compoundsdisclosed herein in connection with embodiments of the present inventionmay exist in multiple crystalline or amorphous forms.

As used herein, the term “isolated,” when used in the context of anisolated compound, antibody, conjugate, etc., refers to a compound ofinterest (e.g., a compound as described herein, a conjugate as describedherein, an antibody as described herein, etc.) that is in an environmentdifferent from that in which the compound naturally occurs. “Isolated”is meant to include compounds of interest (e.g., a compound as describedherein, a conjugate as described herein, or an antibody as describedherein) that are within samples that are substantially enriched for thecompound of interest and/or in which the compound of interest ispartially or substantially purified. As used herein, the term“substantially pure” refers to a compound of interest that is removedfrom its natural environment and is at least 60% free, at least about75% free, at least about 90% free, at least about 95% free, at leastabout 98% free, or more than 98% free, from other components with whichit is naturally associated, and/or with it may be associated duringsynthesis or production.

Certain compounds disclosed herein in connection with embodiments of thepresent invention possess asymmetric carbon atoms (optical centers) ordouble bonds; the racemates, diastereomers, geometric isomers andindividual isomers are encompassed within the scope of the presentinvention.

The compounds may be prepared as a single isomer (e.g., enantiomer,cis-trans, positional, diastereomer) or as a mixture of isomers. In oneembodiment, the compounds are prepared as substantially a single isomer.Methods of preparing substantially isomerically pure compounds are knownin the art. For example, enantiomerically enriched mixtures and pureenantiomeric compounds can be prepared by using synthetic intermediatesthat are enantiomerically pure in combination with reactions that eitherleave the stereochemistry at a chiral center unchanged or result in itscomplete inversion. Alternatively, the final product or intermediatesalong the synthetic route can be resolved into a single stereoisomer.Techniques for inverting or leaving unchanged a particular stereocenter,and those for resolving mixtures of stereoisomers are well known in theart and it is well within the ability of one of skill in the art tochoose and appropriate method for a particular situation. See,generally, Furniss et al. (eds.), Vogel's Encyclopedia of PracticalOrganic Chemistry, 5th ed., Longman Scientific and Technical Ltd.,Essex, 1991, pp. 809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to alsorecite—OCH₂—. Use of a single dash (“-”) or double dash (“-” or “--”)refers to a single covalent bond, while use of “═” refers to a doublebond. The symbol,)₂ or ₂(when displayed with —S, indicates that thecompound inside the parenthesis may be present as a dimer forming adisulfide bond. The dimer may be reduced to a monomer.

The term “acyl” or “alkanoyl” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof, havingthe stated number of carbon atoms and an acyl radical on at least oneterminus of the alkane radical. The “acyl radical” is the group derivedfrom a carboxylic acid by removing the —OH moiety therefrom.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include divalent(“alkylene”) and multivalent radicals, having the number of carbon atomsdesignated (i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.”, where “heteroalkyl” refers to carbon chains having oneor more substitutions at one or more carbon atoms of the hydrocarbonchain fragment. Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl”. Exemplary alkyl groups include those containingbetween about one and about twenty five carbon atoms (e.g. methyl, ethyland the like).

The term “lower alkyl” generally refers to a straight, branched, orcyclic hydrocarbon chain containing 8 or fewer carbon atoms, and cancontain from 1 to 8, from 1 to 6, or from 1 to 4 carbon atoms. Exemplary“lower alkyl” groups include methyl, ethyl, n-propyl, isopropyl,n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl and thelike. “Lower alkyls” can be optionally substituted at one or more carbonatoms of the hydrocarbon chain.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused to refer to those alkyl groups attached to the remainder of themolecule via an oxygen atom, an amino group, or a sulfur atom,respectively.

By “heteroatom” is meant atoms other than a carbon which may be presentin a carbon backbone or a linear, branched or cyclic compound. Exemplaryheteroatoms include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P)and silicon (Si). Heteroatoms can be present in their reduced forms,e.g., —OH, —NH, and —SH.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a straight or branched chain, or cycliccarbon-containing radical, or combinations thereof, having the statednumber of carbon atoms and at least one heteroatom which can be a memberselected from O, N, Si, P and S, wherein the nitrogen, phosphorous andsulfur atoms are optionally oxidized, and the nitrogen heteroatom isoptionally be quaternized. Normally heteroalkyl groups contain no morethan two heteroatoms linked in sequence. The heteroatom(s) O, N, P, Sand Si may be placed at any interior position of the heteroalkyl groupor at the position at which the alkyl group is attached to the remainderof the molecule. Examples include, but are not limited to,—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Generally, up to two heteroatomsmay be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃.

Similarly, the term “heteroalkylene” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic moiety that can be a single ring or multiple rings (usuallyfrom 1 to 3 rings), which are fused together or linked covalently. Theterm “heteroaryl” refers to aryl groups (or rings) that contain from oneto four heteroatoms which are members selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl,2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedbelow.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Exemplary substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ where eachcan be independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ can beindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. In theschemes that follow, the symbol X represents “R” as described above.

The term “amino” or “amine group” refers to the group —NR′R″ (orN⁺RR′R″) where R, R′ and R″ are independently selected from hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl,substituted aryl alkyl, heteroaryl, and substituted heteroaryl. Asubstituted amine is an amine group wherein R′ or R″ is other thanhydrogen. In a primary amino group, both R′ and R″ are hydrogen, whereasin a secondary amino group, either, but not both, R′ or R″ is hydrogen.In addition, the terms “amine” and “amino” can include protonated andquaternized versions of nitrogen, comprising the group —N⁺RR′R″ and itsbiologically compatible anionic counterions.

The term “conjugate” refers to a molecule comprised of two or moremoieties bound together, optionally through a linking group, to form asingle covalent structure. The binding can be made either by a directchemical bond between the components or by use of a linking group. Forexample, a gabapentin conjugate generally refers to a chemical compoundcomposed of a gabapentin derivative covalently bound to a moiety ofinterest, which may be optionally linked through a linking group. Inanother example, a “gabapentin-enzyme conjugate” refers a gabapentinconjugate having an enzyme as the moiety of interest.

A “hapten” generally refers to a small molecule that can be specificallybound by an antibody but cannot induce detectable or significantformation of antibodies unless bound to a carrier protein or other largeantigenic molecule. In the context of the present disclosure, gabapentinand gabapentin derivative is an exemplary hapten. In contrast, an“antigen” refers to a compound that is capable of stimulating an immuneresponse.

The term “linker” as used in the present disclosure refers to a chemicalmoiety that connects at least two substructures of a compound, e.g., toprovide for covalent connection between a gabapentin hapten and a moietyof interest (e.g., a carrier or detectable label).

A “carrier” or “immunogenic carrier,” as the terms are used herein, isan immunogenic substance, commonly a polypeptide, that can join with ahapten (such as a gabapentin moiety), thereby enabling the happen toinduce an immune response and elicit the production of antibodies thatcan bind specifically with the antigen (hapten). Carrier substancesinclude, but are not necessarily limited to, proteins, glycoproteins,complex polysaccharides, particles, and nucleic acids that arerecognized as foreign and thereby elicit an immunologic response fromthe host.

“Polypeptide” as used herein is meant to encompass a polyaminoacid ofany length, and encompasses proteins, protein fragments and peptides.Polypeptides may be genetically encoded or synthetically produced.Polypeptides may also be modified, e.g., by post-translational and/orchemical modification(s).

As used herein, a “detectable label” generally refers to an identifyingtag that can provide for a detectable signal, e.g., luminescence (e.g.,photoluminescence (e.g., fluorescence, phosphorescence),chemoluminescence (e.g., bioluminescence)), radioactivity,immunodetection, enzymatic activity, and the like).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen.

The monoclonal antibodies include hybrid and recombinant antibodiesproduced by splicing a variable (including hypervariable) domain of ananti-inhibitor antibody with a constant domain, or a light chain with aheavy chain, or a chain from one species with a chain from anotherspecies, or fusions with heterologous proteins, regardless of species oforigin or immunoglobulin class or subclass designation, as well asantibody fragments, e.g., Fab, F(ab)2, and Fv!, so long as they exhibitthe desired biological activity. For example, the monoclonal antibodiesmay be made by the hybridoma method first described by Kohler &Milstein, Nature, 256:495 (1975), or may be made by recombinant DNAmethods (U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may alsobe isolated from phage libraries generated using the techniquesdescribed in, e.g., McCafferty et al., Nature, 348:552-554 (1990).

The term “anti-gabapentin antibody” refers to antibodies that arecapable of specifically binding a gabapentin epitope of gabapentin, agabapentin derivative, or a gabapentin conjugate. “Anti-gabapentinantibodies” include both polyclonal and monoclonal antibodies, as wellas antigen-binding fragments thereof as defined above. A “gabapentinepitope” refers to an epitope that is present in gabapentin and in agabapentin derivative (e.g., a gabapentin conjugate).

The term “binds specifically” or “specifically binds” in the context ofantibody binding, refers to high avidity and/or high affinity binding ofan antibody to a specific antigen, e.g., to gabapentin. In specificbinding under appropriate conditions, antibody binding to gabapentin isstronger than binding of the same antibody to any other epitope,particularly those which may be present in molecules in associationwith, or in the same sample, as the gabapentin to be detected, e.g.,binds more strongly (e.g., higher affinity, higher avidity, or both) togabapentin than to a non-gabapentin epitope so that by adjusting bindingconditions the antibody binds almost exclusively to gabapentin, and notto non-gabapentin moieties that may be present in the sample. Antibodieswhich bind specifically to gabapentin may be capable of binding otherantigens at a weak, yet detectable, level (e.g., 10% or less of thebinding shown to gabapentin). Such weak binding, or background binding,is readily discernible from the specific antibody binding to gabapentin,e.g., by use of appropriate controls. “Antibody activity” or “antibodybinding activity” in the context of analyte binding assays generallyrefers to the ability of an antibody to bind a specific antigen inpreference to other potential antigens via the antigen combining sitelocated within a variable region of an immunoglobulin.

By “detectably labeled antibody” an antibody (which, as defined above,includes antigen-binding fragments, etc.) having an attached detectablelabel. The detectable label may be attached by chemical conjugation, butwhere the label is a polypeptide, it could alternatively be attached bygenetic engineering techniques. Methods for production of detectablylabeled proteins are well known in the art. Detectable labels may beselected from a variety of such labels known in the art, but normallyare radioisotopes, fluorophores, enzymes (e.g., horseradish peroxidase),or other moieties or compounds which either emit a detectable signal(e.g., radioactivity, fluorescence, color) or emit a detectable signalafter exposure of the label to its substrate. Various detectablelabel/substrate pairs (e.g., horseradish peroxidase/diaminobenzidine,avidin/streptavidin, luciferase/luciferin), methods for labelingantibodies, and methods for using labeled antibodies to detect anantigen are well known in the art.

“Antibody complex”, “antibody-antigen complex” generally refers to acomplex that results following specific binding of an antibody and itsantigen, e.g., between an anti-gabapentin antibody and gabapentin (or agabapentin derivative, e.g., gabapentin conjugate).

The term “analyte” refers to a substance to be detected in a sample,e.g., gabapentin.

The term “assessing” includes any form of measurement, and includesdetermining the presence or absence if an element. The terms“assessing”, “determining” (e.g., as in “determining the presence orabsence of”), “measuring”, “evaluating”, and “assaying” are usedinterchangeably and include quantitative and qualitative determinations.Assessing may be relative or absolute. “Assessing the presence of”includes determining the amount of something present, and/or determiningwhether it is present or absent. As used herein, the terms“determining,” “measuring,” and “assessing,” and “assaying” are usedinterchangeably and include both quantitative and qualitativedeterminations.

Gabapentin Derivatives

Gabapentin derivatives for the use in gabapentin therapeutic drugmonitoring (TDM) are provided in the present disclosure. The term“gabapentin derivatives” is meant to encompass gabapentin-conjugatesincluding immunogens and assay reagents (e.g., detectably labeledconjugates, such as enzyme conjugates, immobilized conjugates, and thelike), as well as intermediates useful in production of such gabapentinconjugates. In general, a gabapentin derivative is able to compete withgabapentin for binding to an anti-gabapentin antibody, e.g., in agabapentin TDM assay. A schematic representation of the structure ofgabapentin is shown in FIG. 1

Structures of Gabapentin Derivatives

Gabapentin derivatives of the present disclosure can be described ashaving the general formula:

wherein R₁, R₂, or R₃ is —X—W-L-Z, wherein

-   -   when R₁ is —X—W-L-Z, X is NH, and R₂ is —OH, and R₃ is —H,    -   when R₂ is —X—W-L-Z, X is NH, R₁ is —NH₂, and R₃ is —H, and    -   when R₃ is —X—W-L-Z, X is a heteroatom or lower alkyl group, R₁        is —NH₂, and R₂ is —OH, and        W is a lower alkyl group or a carbonyl group;        L is a linker or at least one bond between W and Z; and        Z is H, an alkyl group, a reactive functional group capable of        reacting with a reactive partner to form a covalent bond, or a        moiety of interest;        or a salt thereof.

As noted above, the term “linker” as used in the present disclosurerefers to a chemical moiety that connects at least two substructures ofa compound, e.g., to provide for covalent connection between agabapentin hapten and a moiety of interest (e.g., a carrier ordetectable label). In the context of a gabapentin conjugate, the linkercan be a chemical moiety that is the production of a reaction between areactive functional group and a moiety of interest, e.g., a polypeptide.Exemplary linkers include linear or branched, saturated or unsaturated,hydrocarbon chains of from 1 to 40 carbon atoms, or from 1 to 20 carbonatoms, or from 1 to 10 carbon atoms, which hydrocarbon chains maycontain ring structures (e.g., up to two ring structures) and one ormore heteroatoms.

When W of Formula A is a lower alkyl group, W can be, for example, astraight, branched, or cyclic hydrocarbon chain containing 1 to 8 carbonatoms, from 1 to 6 carbon atoms, or from 1 to 4 carbon atoms.

In general, when W of Formula A is a carbonyl group, then L is a linker,which can be, for example, a hydrocarbon chain of from 1 to 40 carbonatoms, or from 1 to 20 carbon atoms, or from 1 to 10 carbon atoms, whichhydrocarbon chains may optionally contain ring structures (e.g., up totwo ring structures) and one or more heteroatoms.

Where W is a lower alkyl exemplary linkers include:

-   —(CH₂)_(n)C(O)—,-   —C(O)(CH₂)_(n)—,-   —C(O)(CH₂)_(n)NH—C(O)—,-   —C(O)(CH₂)_(m)NH—C(O)(CH₂)_(n)—,-   —(CH₂)_(n)SCH₂C(O)—,-   —(CH₂)_(m)SCH₂C(O)(CH₂)_(n)—,-   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,-   —(CH₂)_(n)NH—C(O)—,-   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—,-   —C(O)—(CH₂)_(n)—, and-   —(CH₂)_(n)—;    wherein m, n, o, and p are independently selected from an integer    from 0 to 10, or from 1 to 10.

Where W is a carbonyl, exemplary linkers include:

-   —(CH₂)_(n)C(O)—,-   —(CH₂)_(n)SCH₂C(O)—,-   —(CH₂)_(m)SCH₂C(O)(CH₂)_(n)—,-   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,-   —(CH₂)_(n)NH—C(O)—,-   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—, and-   —(CH₂)_(n)—;    wherein m, n, o, and p are independently selected from an integer    from 0 to 10, or from 1 to 10.

In some embodiments, such as when Z is a reactive functional group andR1 is —X—W-L-Z, W is a lower alkyl. In some embodiments, such as when Zis a reactive functional group and R2 is —X—W-L-Z, the reactivefunctional group is not OH or COOH.

In some embodiments, such as when Z is a reactive functional group andR1 is X—W-L-Z, and —X—W-L-Z does not include a ring.

In some embodiments, the moiety of interest is not a single amino acidresidue, but may be a polyamino acid chain (i.e., polypeptide). In someembodiments, the moiety of interest is not a bile acid. In someembodiments where the moiety of interest is a polypeptide, thepolypeptide is other than a transporter protein. In some embodiments,the gabapentin derivative is not a bile acid conjugate. In someembodiments, the moiety of interest is not a bile acid.

When Z of Formula A is a reactive functional group capable of reactingwith a reactive partner to form a covalent bond, Z can be, for example ahalogen (e.g., Br, Cl, I, and the like), OH, SH, NH₂, O-lower alkyl,epoxy, S-acyl, carboxyl, maleimidyl, haloacetamide, hydroxysuccinimidyl,succinimidyl, carbonate, anhydride, imidate, isocyanates,isothiocyanates, imidoesters, maleimides, thiolactones, diazoniumgroups, acrylamide, an acyl azide, an acyl nitrile, an alkyl halide, ananiline, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a halotriazine, a hydrazine, a hydrazide, an imido ester, aphosphoramidite, a reactive platinum complex, a sulfonyl halide, and aphotoactivatable group.

Moieties of interest for Z of Formula A include immunogenic carriers(e.g., carrier proteins) and detectable labels. Moieties of interest aredescribed in more detail below.

Salts of gabapentin derivatives include, but are not necessarily limitedto, alkali metal salts, such as (sodium salts, potassium salts,magnesium salts), halide salts (e.g., bromo, chloro, and the like);acetate salts (e.g., salts with trifluoroacetic acid, and the like).

Gabapentin derivatives can be further described as having a formulaselected from

wherein the wavy line (“

”) indicates the point at which the gabapentin moiety is attached to theremainder of the gabapentin derivative, e.g., attached through one ormore chemical moieties to a moiety of interest, e.g., a gabapentinconjugate, e.g., a gabapentin conjugate having a immunogenic carrier ora detectable label. For example, the wavy line can represent a site ofattachment to a polypeptide, detectable label, solid support, and thelike, where attachment can be through a chemical structure as set outabove.

Gabapentin derivatives may be provided as dimers. Such dimericgabapentin derivatives can form from reaction of gabapentin derivativeshaving a sulfhydryl group or bromoacetyl group as a reactive functionalgroup (Z). Thus, for example, dimerized gabapentin derivatives connectedthrough a disulfide bond of a reactive group. Such dimers may be reducedto result in two monomeric gabapentin derivatives.

Further examples of gabapentin derivatives are described below.

Gabapetin Derivatives of Formula I

In one embodiment the gabapentin derivative has Formula I below, inwhich the gabapentin derivative includes an extension from the aminegroup of gabapentin or its salt:

W, L, and Z can be as defined above in Formula A, and can be anycombinations exemplified for Formula A as set out above. Accordingly,when W of Formula I is a lower alkyl group, W can be, for example, astraight, branched, or cyclic hydrocarbon chain containing 1 to 8 carbonatoms, from 1 to 6 carbon atoms, or from 1 to 4 carbon atoms. Ingeneral, when W of Formula I is a carbonyl group, then L is a linker,which can be, for example, a hydrocarbon chain of from 1 to 40 carbonatoms, or from 1 to 20 carbon atoms, or from 1 to 10 carbon atoms andone or more heteroatoms.

Where W is a lower alkyl exemplary linkers for gabapentin derivatives ofFormula I include:

-   —(CH₂)_(n)C(O)—,-   —C(O)(CH₂)_(n)—,-   —C(O)(CH₂)_(n)NH—C(O)—,-   —C(O)(CH₂)_(m)NH—C(O)(CH₂)_(n)—,-   —(CH₂)_(n)SCH₂C(O)—,-   —(CH₂)_(m)SCH₂C(O)(CH₂)_(n)—,-   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,-   —(CH₂)_(n)NH—C(O)—,-   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—,-   —C(O)—(CH)_(n)—, and-   —(CH₂)_(n)—;    wherein m, n, o, and p are independently selected from an integer    from 0 to 10, or from 1 to 10.

Where W is a carbonyl, exemplary linkers include:

-   —(CH₂)_(n)C(O)—,-   —(CH₂)_(n)SCH₂C(O)—,-   —(CH₂)_(m)SCH₂C(O)(CH₂)_(n)—,-   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,-   —(CH₂)_(n)NH—C(O)—,-   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—, and-   —(CH₂)_(n)—;    wherein m, n, o, and p are independently selected from an integer    from 0 to 10, or from 1 to 10.

When Z of Formula A is a reactive functional group capable of reactingwith a reactive partner to form a covalent bond, Z can be, for example ahalogen (e.g., Br, Cl, I, and the like), OH, SH, NH₂, O-lower alkyl,epoxy, S-acyl, carboxyl, maleimidyl, haloacetamide, hydroxysuccinimidyl,succinimidyl, carbonate, anhydride, imidate, isocyanates,isothiocyanates, imidoesters, maleimides, thiolactones, diazoniumgroups, acrylamide, an acyl azide, an acyl nitrile, an alkyl halide, ananiline, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a halotriazine, a hydrazine, a hydrazide, an imido ester, aphosphoramidite, a reactive platinum complex, a sulfonyl halide, and aphotoactivatable group. Reactive functional groups for Z can be any ofthose as exemplified for Z of Formula A.

Moieties of interest for Z of Formula I include immunogenic carriers(e.g., carrier proteins) and detectable labels. Moieties of interest aredescribed in more detail below.

In specific embodiments, —W-L-Z of Formula I may be —(CH₂)_(m)—SH,—CO—(CH₂)_(n)—Br, or —(CH₂)_(o)—S—CH₂—CO—CH₂—Br, wherein m, n, and o areindependently selected from 2 or 3.

Exemplary gabapentin derivatives of Formula I are provided in FIG. 2 ascompounds Gabapentin I, II, III, IV, and XIV. In reduced gabapentin I, Wand L are methyl and Z is SH (or may also be described per Formula Iwherein L is a bond between W and Z, and W is ethyl). Gabapentin I mayexist as a dimer forming a disulfide bond through the thiol groups asshown in FIG. 2. In gabapentin II, W is methyl, L is ethyl, and Z is SH.In gabapentin III, W is methyl, L is (CH₂)SCH₂C(O)CH₂, and Z is Br. Ingabapentin IV, W is carbonyl, L is methyl, and Z is Br. In gabapentinXIV, W is methyl, L is —(CH₂)₂NH—C(O)(CH₂)—, and Z is Br.

Salts of gabapentin derivative of Formula I can be those as describedabove for Formula A.

Gabapetin Derivatives of Formula II

In another embodiment, gabapentin derivatives are characterized by anextension from the carbonyl group of gabapentin or its salt, which canbe described by the formula:

W, L, and Z can be as defined above in Formula A, and can be anycombinations exemplified for Formula A as set out above. Accordingly,when W of Formula II is a lower alkyl group, W can be, for example, astraight, branched, or cyclic hydrocarbon chain containing 1 to 8 carbonatoms, from 1 to 6 carbon atoms, or from 1 to 4 carbon atoms. Ingeneral, when W of Formula A is a carbonyl group, then L is a linker,which can be, for example, a hydrocarbon chain of from 1 to 40 carbonatoms, or from 1 to 20 carbon atoms, or from 1 to 10 carbon atoms, whichhydrocarbon chains may optionally contain ring structures (e.g., up totwo ring structures) and one or more heteroatoms.

Where W is a lower alkyl exemplary linkers for gabapentin derivatives ofFormula II include:

-   —(CH₂)_(n)C(O)—,-   —C(O)(CH₂)_(n)—,-   —C(O)(CH₂)_(n)NH—C(O)—,-   —C(O)(CH₂)_(m)NH—C(O)(CH₂)_(n)—,-   —(CH₂)_(n)SCH₂C(O)—,-   —(CH₂)_(m)SCH₂C(O)(CH₂)_(n)—,-   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,-   —(CH₂)_(n)NH—C(O)—,-   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—,-   —C(O)—(CH)_(n)—, and-   —(CH₂)_(n)—;    wherein m, n, o, and p are independently selected from an integer    from 0 to 10, or from 1 to 10.

Where W is a carbonyl, exemplary linkers include:

-   —(CH₂)_(n)C(O)—,-   —(CH₂)_(n)SCH₂C(O)—,-   —(CH₂)_(m)SCH₂C(O)(CH₂)_(n)—,-   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,-   —(CH₂)_(n)NH—C(O)—,-   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—, and-   —(CH₂)_(n)—;    wherein m, n, o, and p are independently selected from an integer    from 0 to 10, or from 1 to 10.

When Z of Formula A is a reactive functional group capable of reactingwith a reactive partner to form a covalent bond, Z can be, for example ahalogen (e.g., Br, Cl, I, and the like), OH, SH, NH₂, O-lower alkyl,epoxy, S-acyl, carboxyl, maleimidyl, haloacetamide, hydroxysuccinimidyl,succinimidyl, carbonate, anhydride, imidate, isocyanates,isothiocyanates, imidoesters, maleimides, thiolactones, diazoniumgroups, acrylamide, an acyl azide, an acyl nitrile, an alkyl halide, ananiline, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a halotriazine, a hydrazine, a hydrazide, an imido ester, aphosphoramidite, a reactive platinum complex, a sulfonyl halide, and aphotoactivatable group. Reactive functional groups for Z can be any ofthose as exemplified for Z of Formula A.

Moieties of interest for Z of Formula II include immunogenic carriers(e.g., carrier proteins) and detectable labels. Moieties of interest aredescribed in more detail below.

In exemplary embodiments, —W-L-Z of Formula II may be —(CH₂)_(m)—SH,—CO—(CH₂)_(n)—Br, or —(CH₂)_(o)—S—CH₂—CO—CH₂—Br, wherein m, n, and o areindependently selected from 2 or 3.

Exemplary gabapentin derivatives of Formula II are provided in FIG. 2.In reduced gabapentin V, W is methyl, L is ethyl and Z is SH. In reducedgabapentin VI, W and L are methyl and Z is SH. Gabapentin VI may beprovided as a dimer forming a disulfide bond through the thiol groups.In gabapentin VII, W is methyl, L is (CH₂)SCH₂C(O)CH₂, and Z is Br.

Salts of gabapentin derivative of Formula II can be those as describedabove for Formula A.

Gabapetin Derivatives of Formula III

In a further embodiment the gabapentin derivatives are characterized ashaving an extension from carbon 4 of gabapentin. Such gabapentinderivatives can be described by the formula:

wherein X is a heteroatom or lower alkyl group. For example, X can beNH, S, SH, O, or OH. When X is a lower alkyl group, X can be, forexample, a straight, branched, or cyclic hydrocarbon chain containing 1to 8 carbon atoms, from 1 to 6 carbon atoms, or from 1 to 4 carbonatoms.

W, L, and Z of Formula III can be as defined above in Formula A, and canbe any combinations exemplified for Formula A as set out above.Accordingly, when W of Formula III is a lower alkyl group, W can be, forexample, a straight, branched, or cyclic hydrocarbon chain containing 1to 8 carbon atoms, from 1 to 6 carbon atoms, or from 1 to 4 carbonatoms. In general, when W of Formula A is a carbonyl group, then L is alinker, which can be, for example, a hydrocarbon chain of from 1 to 40carbon atoms, or from 1 to 20 carbon atoms, or from 1 to 10 carbonatoms, which hydrocarbon chains may optionally contain ring structures(e.g., up to two ring structures) and one or more heteroatoms.

Where W is a lower alkyl exemplary linkers for gabapentin derivatives ofFormula III include:

-   —(CH₂)_(n)C(O)—,-   —C(O)(CH₂)_(n)—,-   —C(O)(CH₂)_(n)NH—C(O)—,-   —C(O)(CH₂)_(m)NH—C(O)(CH₂)_(n)—,-   —(CH₂)_(n)SCH₂C(O)—,-   —(CH₂)_(m)SCH₂C(O)(CH₂)_(n)—,-   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,-   —(CH₂)_(n)NH—C(O)—,-   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—,-   —C(O)—(CH₂)_(n)—, and-   —(CH₂)_(n)—;    wherein m, n, o, and p are independently selected from an integer    from 0 to 10, or from 1 to 10.

Where W is a carbonyl, exemplary linkers include:

-   —(CH₂)_(n)C(O)—,-   —(CH₂)_(n)SCH₂C(O)—,-   —(CH₂)_(m)SCH₂C(O)(CH₂)_(n)—,-   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,-   —(CH₂)_(n)NH—C(O)—,-   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—, and-   —(CH₂)_(n)—;    wherein m, n, o, and p are independently selected from an integer    from 0 to 10, or from 1 to 10.

When Z of Formula A is a reactive functional group capable of reactingwith a reactive partner to form a covalent bond, Z can be, for example ahalogen (e.g., Br, Cl, I, and the like), OH, SH, NH₂, O-lower alkyl,epoxy, S-acyl, carboxyl, maleimidyl, haloacetamide, hydroxysuccinimidyl,succinimidyl, carbonate, anhydride, imidate, isocyanates,isothiocyanates, imidoesters, maleimides, thiolactones, diazoniumgroups, acrylamide, an acyl azide, an acyl nitrile, an alkyl halide, ananiline, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a halotriazine, a hydrazine, a hydrazide, an imido ester, aphosphoramidite, a reactive platinum complex, a sulfonyl halide, and aphotoactivatable group. Reactive functional groups for Z can be any ofthose as exemplified for Z of Formula A. In specific embodiments, —W-L-Zof Formula III may be —(CH₂)_(m)—SH, —CO—(CH₂)_(n)—Br, or—(CH₂)_(o)—S—CH₂—CO—CH₂—Br, wherein m, n, and o are independentlyselected from 2 or 3.

Moieties of interest for Z of Formula III include immunogenic carriers(e.g., carrier proteins) and detectable labels. Moieties of interest aredescribed in more detail below.

In exemplary gabapentin derivatives of Formula III, —X—W-L-Z may be—NH—(CH₂)_(m)—SH, —NH—CO—(CH₂)_(n)—Br, and—NH—(CH₂)_(o)—S—CH₂—CO—CH₂—Br, wherein m, n, and o are independentlyselected from 2 or 3.

Exemplary gabapentin derivatives of Formula III (gabapentin VIII, IX, X,XI, XII and XIII) are provided in FIG. 2. In reduced gabapentin VIII, Xis NH, W is methyl, L is (CH₂)₂, and Z is SH. Gabapentin VIII may beprovided as a dimer forming a disulfide bond through the thiol groups asshown in FIG. 2. In gabapentin IX, L is NH, W is methyl, L is(CH₂)₂SCH₂C(O)CH₂, and Z is Br. In gabapentin X, the moiety X is NH, Wand L are methyl, and Z is SH. In gabapentin XI, X is NH, W is carbonyl,L is methyl, and Z is Br. In gabapentin XII, X is NH, W is methyl, L is—(CH₂)NH—C(O)(CH₂)—, and Z is Br. In gabapentin XIII, X and W aremethyl, L is —NH—C(O)(CH₂)—, and Z is Br.

Salts of gabapentin derivative of Formula III can be those as describedabove for Formula A.

Methods of Making Gabapentin Derivatives

The compounds of the invention are synthesized by an appropriatecombination of generally well known synthetic methods. Techniques usefulin synthesizing the compounds of the invention are both readily apparentand accessible to those of skill in the relevant art. The discussionbelow is offered to illustrate certain of the diverse methods availablefor use in assembling the compounds of the invention; it is not intendedto define the scope of reactions or reaction sequences that are usefulin preparing the compounds of the present invention.

In Schemes 1-5, preparatory schemes for 4-amino gabapentin derivatives(formula III) are presented.

In scheme 1, compound 1 is reacted as described in the literature U.S.Pat. Publication No. 2007/0123591 and Chinese Pat. CN1740161, which areincorporated herein by reference, to form compound 2. In the next stepof the reaction scheme, the amine of compound 2 is protected with anN-tert-butoxycarbonyl (t-BOC) protecting group to form compound 3.Compound 3 is then oxidized with ozone in an ozonolysis reaction to formthe aldehyde compound 4. In the next step, compound 4 is reacted withammonia (NH₃) and sodium cyanoborohydride (NaCNBH₃) to form aminecompound 5. In the next step, compound 5 is bromoacetylated with bromoacetyl NHS ester, and then the t-BOC protecting group is removed withhydrogen bromide (HBr) to form compound 6, which is a4-[2-(2-bromo-acetylamino)-ethyl]derivative of gabapentin.

In Scheme 2, compound 7 is reacted as described in the literature U.S.Pat. Publication No. 2007/0123591 and Chinese Pat. CN1740161, which areincorporated herein by reference, to form compound 8. In the next stepof the reaction scheme, the amine of compound 8 is protected with at-BOC protecting group to form compound 9. Compound 9 is then oxidizedwith ozone in an ozonolysis reaction to form compound 10. In the nextstep, compound 10 is reacted with ammonia (NH₃) and sodiumcyanoborohydride (NaCNBH₃) to form amine compound 11. In the next step,compound 11 is bromoacetylated with bromo acetyl NHS ester, and then thet-BOC protecting group is removed with hydrogen bromide (HBr) to formcompound 12, which is a 4-(2-bromo-acetylamino) derivative ofgabapentin.

In Scheme 3, compound 10 is reacted with ethylenediamine and sodiumcyanoborohydride (NaCNBH₃) to form amine compound 13. In the next stepof the reaction scheme, compound 13 is bromoacetylated with bromo acetylNHS ester, and then the t-BOC protecting group is removed with hydrogenbromide (HBr) to form compound 14, which is a4-[2-(2-bromo-acetylamino)-ethylamino]derivative of gabapentin.

In Scheme 4, compound 10 is reacted with2-(2-Amino-ethyldisulfanyl)-ethylamine and sodium cyanoborohydride(NaCNBH₃) to form amine compound 15. In the next step of the reactionscheme, the t-BOC protecting group of compound 15 is removed withhydrogen bromide (HBr) to form compound 16, which is a4-(2-mercapto-ethylamino) derivative of gabapentin.

In Scheme 5, compound 10 is reacted with3-(3-Amino-propyldisulfanyl)-propylamine and sodium cyanoborohydride(NaCNBH₃) to form amine compound 17. In the next step of the reactionscheme, the t-BOC protecting group of compound 17 is removed withhydrogen bromide (HBr) to form compound 18, which is a4-(3-mercapto-propylamino) derivative of gabapentin.

In Schemes 6-7, preparatory schemes for carboxyl modified derivatives ofgabapentin (formula II) are presented.

In Scheme 6, compound 19 is reacted with2-(2-Amino-ethyldisulfanyl)-ethylamine to form compound 20. In the nextstep of the reaction scheme, compound 20 is deprotected with HCl to fromcompound 21, which is a carboxyl modified derivative of gabapentin.

In Scheme 7, compound 19 is reacted with3-(3-Amino-propyldisulfanyl)-propylamine to form compound 23. In thenext step of the reaction scheme, compound 23 is deprotected with HCl tofrom compound 24, which is a carboxyl modified derivative of gabapentin.

In Schemes 8-11, preparatory schemes for amine modified derivatives ofgabapentin are presented.

In Scheme 8, gabapentin is reacted with bromo acetyl NHS ester to formcompound 25, which is a 2-bromo-acetylamino derivative of gabapentin.

In Scheme 9, compound 26 is reacted with ethylenediamine to form aminecompound 27. In the next step of the reaction scheme, compound 27 isreacted with bromoacetyl NHS ester to form a t-butyl ester intermediate(not shown), which is subsequently deprotected with trifluoroacetic acid(TFA) to form compound 28, which is a3-(2-bromo-acetylamino)-propylamino derivative of gabapentin.

In Scheme 10, compound 26 is reacted with2-(2-Amino-ethyldisulfanyl)-ethylamine to form the t-butyl estercompound 29. In the next step of the reaction scheme, compound 29 isdeprotected with TFA to form compound 30, which is a2-mercapto-ethylamino derivative of gabapentin.

In Scheme 11, compound 26 is reacted with3-(3-Amino-propyldisulfanyl)-propylamine to form the t-butyl estercompound 31. In the next step of the reaction scheme, compound 31 isdeprotected with TFA to form compound 32, which is a3-mercapto-propylamino derivative of gabapentin.

Gabapentin Conjugates

A gabapentin conjugate includes a gabapentin moiety and covalently boundmoiety of interest, wherein the gabapentin moiety and moiety of interestcan be covalently bound as a result of reaction through a reactivefunctional group of the gabapentin derivative.

Gabapentin conjugates of the present disclosure are thus of the generalformula:

wherein R₁, R₂, or R₃ is —X—W-L-Z, wherein

-   -   when R₁ is —X—W-L-Z, X is NH, and R₂ is —OH, and R₃ is —H,    -   when R₂ is —X—W-L-Z, X is NH, R₁ is —NH₂, and R₃ is —H, and    -   when R₃ is —X—W-L-Z, X is a heteroatom or lower alkyl group, R₁        is —NH₂, and R₂ is —OH, and        W is a lower alkyl group or a carbonyl group;        L is a linker or at least one bond between W and Z; and        Z is a moiety of interest (e.g., an immunogenic carrier or        detectable label);        or a salt thereof.

Accordingly, when W of Formula A′ is a lower alkyl group, W can be, forexample, a straight, branched, or cyclic hydrocarbon chain containing 1to 8 carbon atoms, from 1 to 6 carbon atoms, or from 1 to 4 carbonatoms. In general, when W of Formula A′ is a carbonyl group, then L is alinker, which can be, for example, a hydrocarbon chain of from 1 to 40carbon atoms, or from 1 to 20 carbon atoms, or from 1 to 10 carbonatoms, which hydrocarbon chains may optionally contain ring structures(e.g., up to two ring structures) and one or more heteroatoms.

Where W is a lower alkyl exemplary linkers for gabapentin derivatives ofFormula A′ include:

-   —(CH₂)_(n)C(O)—,-   —C(O)(CH₂)_(n)—,-   —C(O)(CH₂)_(n)NH—C(O)—,-   —C(O)(CH₂)_(m)NH—C(O)(CH₂)_(n)—,-   —(CH₂)_(n)SCH₂C(O)—,-   —(CH₂)_(m)SCH₂C(O)(CH₂)_(n)—,-   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,-   —(CH₂)_(n)NH—C(O)—,-   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—,-   —C(O)—(CH)_(n)—, and-   —(CH₂)_(n)—;    wherein m, n, o, and p are independently selected from an integer    from 0 to 10, or from 1 to 10.

Where W is a carbonyl, exemplary linkers include:

-   —(CH₂)_(n)C(O)—,-   —(CH₂)_(n)SCH₂C(O)—,-   —(CH₂)_(m)SCH₂C(O)(CH₂)_(n)—,-   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,-   —(CH₂)_(n)NH—C(O)—,-   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—, and-   —(CH₂)_(n)—;    wherein m, n, o, and p are independently selected from an integer    from 0 to 10, or from 1 to 10.

Salts of gabapentin derivatives include, but are not necessarily limitedto, alkali metal salts, such as (sodium salts, potassium salts,magnesium salts), halide salts (e.g., bromo, chloro, and the like);acetate salts (e.g., salts with trifluoroacetic acid, and the like).

In one embodiment, the moiety of interest is bound to a gabapentinconjugate through a linker selected from CONH, NHCO, NHCONH,NH—(C═S)—NH, O—CO—NH, NH—O—CO, S, NH—(C═NH), N═N, or NH.

The present disclosure also provides gabapentin conjugates of theformula:

wherein W and L are as defined in Formula A′, and Z is a moiety ofinterest.

Accordingly, exemplary linkers for gabapentin conjugates of Formula I′are as listed above in Formula A′. Salts of gabapentin conjugate ofFormula A′ can include those as described for Formula A′.

Exemplary gabapentin conjugates of Formula I′ are provided in FIG. 3 asConjugates I, II, and III. In Conjugate I W is a lower alkyl (e.g.,(CH₂)₂), and L is —S—(CH₂)(C═O)— which is joined to a carrier protein(Z) through an amine of an amino acid residue of the protein(exemplified by keyhole limpet hemocyanin (KLH)). In Conjugate II, W isa carbonyl, L is a methyl group, and Z is a carrier protein (exemplifiedby KLH) which is attached to the gabapentin moiety through a sulfhydrylgroup of a cysteine residue of the protein. In Conjugate III, W is alower alkyl (e.g., (CH₂)₂), and L is —S—(CH₂)(C═O)(CH₂)—, and Z is acarrier protein (e.g., KLH) which is attached to the gabapentin moietythrough a sulfhydryl group of a cysteine residue of the protein.

In another embodiment, gabapentin conjugates are characterized by anextension from the carbonyl group of gabapentin, which can be describedby the formula:

wherein W and L are as defined in Formula A′, and Z is a moiety ofinterest.

Accordingly, exemplary linkers for gabapentin conjugates of Formula II′are as listed above in Formula A′. Salts of gabapentin conjugates ofFormula II′ can be those as described above for Formula A′.

Exemplary gabapentin conjugates of Formula II′ are provided in FIG. 3 asConjugates IV and V. In Conjugate IV, W is a lower alkyl (e.g., (CH₂)₂),L is —S—(CH₂)(C═O)—, and Z is exemplified by a carrier protein (e.g.,KLH) which is linked to the gabapentin moiety through an amine of anamino acid residue of the protein. In Conjugate V, W is a lower alkyl(e.g., (CH₂)₂), L is —S—(CH₂)(C═O)(CH₂)—, and Z is exemplified by acarrier protein (e.g., KLH) which is linked to the gabapentin moietythrough a sulfhydryl of a cysteine residue of the protein.

In a further embodiment the gabapentin conjugates are characterized ashaving an extension from carbon 4 of gabapentin. Such gabapentinconjugates can be described by the formula:

wherein X is a heteroatom or lower alkyl group, W and L are as definedin Formula A′.

Accordingly, exemplary linkers for gabapentin conjugates of Formula III′are as listed above in Formula A′. Salts of gabapentin conjugates ofFormula III′ can be those as described above for Formula A′.

Exemplary gabapentin conjugates of Formula III′ are provided in FIG. 3as Conjugates Vi and VII. In Conjugate VI, W is a lower alkyl (e.g.,—(CH₂)₃—), L is —S—(CH₂)(C═O)—, and Z is exemplified by a carrierprotein (e.g., KLH) which is linked to the gabapentin moiety through anamine of an amino acid residue of the protein. In Conjugate VII, W is alower alkyl (e.g., —(CH₂)₃—), L is —S—(CH₂)(C═O)(CH₂)—, and Z isexemplified by a carrier protein (e.g., KLH) which is linked to thegabapentin moiety through a sulfhydryl of a cysteine residue of theprotein.

Where the moiety of interest has multiple available covalent attachmentsites for a gabapentin moiety (e.g., a reactive partner having multiplereaction sites for reaction with a gabapentin derivative), thegabapentin conjugate can include more than one gabapentin moiety.Accordingly, gabapentin conjugates of the present disclosure includethose in which two or more, gabapentin moieties are bound to the samemoiety of interest (e.g., polypeptide (e.g., carrier protein), solidsupport (e.g., Sepharose® bead, particle (e.g., gold particle, magneticparticle). Such gabapentin conjugates can thus be represented by aformula as follows:

wherein W and L are as defined in Formula A′, and Z is a moiety ofinterest having two or more attachment sites for a gabapentin moiety;

wherein W and L are as defined in Formula A′, and Z is a moiety ofinterest having two or more attachment sites for a gabapentin moiety; or

wherein X is a heteroatom or lower alkyl group, W and L are as definedin Formula A′, and wherein q is at least 1, and may be 2 or more, 5 ormore, or 10 or more, 15 or more, up to a number of reaction sitesavailable on Z. For example, where Z is a polypeptide, q can be up tothe number of accessible amino acid residues reactive with a gabapentinderivative having an appropriate reactive functional group.Moieties of Interest of Gabapentin Conjugates

In general, the moiety of interest of gabapentin conjugates of thepresent disclosure can be any suitable chemical entity or support,especially one adapted for use in an assay, or for generating reagentsuseful in such assays (e.g., anti-gabapentin antibody production),described herein.

Accordingly, the moiety of interest can be, for example, an immunogeniccarrier, a detectable label, or a support.

Exemplary immunogenic carriers include polypeptides (which term is usedto encompass amino acid chains of any length, including peptides andproteins), modified polypeptides (e.g., post-translationally and/orchemically modified, e.g., lipoproteins, glycoproteins, and the like),and polysaccharides, with polypeptide immunogenic carriers being ofparticular interest.

Further exemplary moieties of interest, which may serve as detectablelabels, include polypeptides having an immunodetectable epitope (i.e.,detectable by binding of a binding partner that specifically binds theepitope (e.g., an HA tag)), nucleic acids (which can be detectable byuse of a hybridization probe or by PCR-based methods), radioactiveisotopes, enzymes (including enzyme fragments, enzyme donor fragments,enzyme acceptor fragments, coenzymes), enzyme ligands (e.g., enzymesubstrates, enzyme inhibitors), fluorescent moieties (includingfluorophores and quenchers), phosphorescent moieties, anti-stokesup-regulating moieties, chemiluminescent moieties, luminescent moieties,chromophores, radioactive isotopes, and combinations thereof.

Further exemplary moieties of interest which may serve as a supportinclude, but are not necessarily limited to, solid supports (e.g.,arrays), particles (including gold particles, microparticles, magneticparticles, beads, and the like), and liposomes.

As noted above, a “detectable label” generally refers to an identifyingtag that can provide for a detectable signal, e.g., luminescence (e.g.,photoluminescence (e.g., fluorescence, phosphorescence),chemoluminescence (e.g., bioluminescence)), radioactivity,immunodetection, enzymatic activity, and the like). Examples of a labelinclude a polypeptide such as an antigen, enzyme, an antibody, a nucleicacid, a fluorophor, a quencher (e.g., of a FRET pair), a phosphorescentgroup, a chemiluminescent group, a chromophoric group, anelectrochemically active group, an electrochemiluminescent group, agroup that undergoes a change in fluorescence, phosphorescence,chemiluminescence or electrochemical property upon binding (U.S. Pat.Nos. 6,203,974 and 6,159,750), a solid particle, a gold particle, aradioactive isotope, an enzyme ligand (e.g., an enzyme inhibitor, anenzyme substrate), an enzyme cofactor, a member of an enzymedonor-acceptor pair.

The detectable label can be a “non-isotopic signal-generating moiety.”“Non-isotopic signal-generating moiety”, as used herein, refers to amoiety that does not emit radioactivity as a detectable signal. By wayof example, a non-isotopic signal-generating moiety is an enzyme,fluorescent compound, or a luminescent compound.

Exemplary moieties of interest are further described below.

Gabapentin-Carrier Protein Conjugates

In one embodiment, the moiety of interest is an immunogenic carrier,particularly an immunogenic carrier protein. Such gabapentin conjugatesfind use in production of anti-gabapentin antibodies, which in turn finduse in the gabapentin detection assays described herein. Exemplarycarriers include, but are not necessarily limited to, proteins,glycoproteins, complex polysaccharides, particles, and nucleic acidsthat are recognized as foreign and thereby elicit an immunologicresponse from the host.

Various protein types may be employed as a poly(amino acid) immunogeniccarrier. These types include albumins, serum proteins, e.g., globulins,ocular lens proteins, lipoproteins, etc. Illustrative proteins includebovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), eggovalbumin, bovine gamma-globulin (BGG), etc. Immunogenic polypeptidesinclude genetically-encodable and synthetic polypeptides.

The immunogenic carrier can also be a polysaccharide, which is a highmolecular weight polymer built up by repeated condensations ofmonosaccharides. Examples of polysaccharides are starches, glycogen,cellulose, carbohydrate gums such as gum arabic, agar, and so forth. Thepolysaccharide can also contain polyamino acid residues and/or lipidresidues.

The immunogenic carrier can also be a poly(nucleic acid) either alone orconjugated to one of the above mentioned poly(amino acids) orpolysaccharides. The immunogenic carrier can also be a particle. Theparticles are generally at least about 0.02 microns and not more thanabout 100 micron, and usually about 0.05 micron to 10 micron indiameter. The particle can be organic or inorganic, swellable ornon-swellable, porous or non-porous, optionally of a densityapproximating water, generally from about 0.7 to 1.5 g/mL, and composedof material that can be transparent, partially transparent, or opaque.The particles can be biological materials such as cells andmicroorganisms, including non-limiting examples such as erythrocytes,leukocytes, lymphocytes, hybridomas, Streptococcus, Staphylococcusaureus, E. coli, and viruses. The particles can also be comprised oforganic and inorganic polymers, liposomes, latex, phospholipid vesicles,or lipoproteins.

Immunogenic carriers exemplified herein include keyhole limpethemocyanin (KLH) and bovine serum albumin (BSA). FIG. 3 shows schematicrepresentation of exemplary gabapentin conjugates having a gabapentinmoiety linked to KLH. Dimerized gabapentin conjugates can be linkedthrough a disulfide bond, which may be reduced to generate monomericgabapentin conjugates, e.g., by reaction with DTT or TCEP. Gabapentincarrier protein conjugates may comprise a plurality of gabapentinderivatives covalently bonded to the protein carrier, as discussed inFormulae I′″, II′″, and III′″ above.

Gabapentin-Enzyme Conjugates

In another embodiment, the moiety of interest of the gabapentinconjugate is an enzyme. In general, the enzyme can serve as anon-isotopic signal generating moiety, and may be any enzyme thatprovides for a detectable signal useful in, for example, an immunoassaydescribed herein. Exemplary enzymes include alkaline phosphatase,β-galactosidase, horse radish peroxidase, glucose-6-phosphatedehydrogenase (G6PDH), and the like. The G6PDH refers to both anaturally occurring G6PDH and G6PDH variants that contain one or morecysteine residues non-native to naturally-occurring G6PDH.

In one embodiment of interest, gabapentin enzyme conjugates provide animmunoassay reagent that compete with gabapentin that may be present ina sample for binding to an anti-gabapentin antibody, wherein thepresence of absence of a detectable signal provided by the enzyme isindicative of the presence or absence of a gabapentin-anti-gabapentinantibody complex. Gabapentin enzyme conjugates containing G6PDH(including naturally occurring G6PDH and G6PDH variants, particularlyG6PDH cysteine variants) find particular use in such assays.

FIG. 4 shows schematic representation of exemplary gabapentin enzymeconjugates, exemplified by gabapentin G6PDH conjugates. Dimerizedgabapentin enzyme conjugates can be linked through a disulfide bond,which may be reduced to generate monomeric gabapentin enzyme conjugates,e.g., by reaction with DTT or TCEP. Gabapentin enzyme conjugates maycomprise a plurality of gabapentin derivatives covalently bonded to theprotein carrier, as discussed in Formulae I′″, II′″, and III′″ above.

Other Gabapentin Conjugates

The moiety of interest may be a support, thus immobilizing thegabapentin conjugate. The moiety of interest may provide a detectablesignal, such as a fluorophore, a fluorescence quencher, a radioisotope,and metal particle (e.g., in SERS-based assays). Gabapentin conjugatescan include, for example, a first member of a FRET pair (e.g., a memberof a fluorophore/quencher pair), where the gabapentin conjugate is usedin connection with an anti-gabapentin antibody having the second memberof the FRET pair, such that binding of the labeled anti-gabapentinantibody to the labeled gabapentin conjugate in a complex provides for adetectable signal different from that when the labeled anti-gabapentinantibody and labeled gabapentin conjugate are not in a complex with oneanother (e.g., as when binding of gabapentin blocks binding of theantibody to the gabapentin conjugate).

Methods of Making Gabapentin Conjugates

Gabapentin conjugates are typically prepared by synthesizing agabapentin derivative having a reactive functional group (e.g., asdescribed above), and incubating the gabapentin derivative with areactive partner (e.g., a protein) under conditions that permit aconjugation reaction to occur, and then separating the conjugate fromthe reaction mixture.

For example, a protein conjugate can be prepared by combining an excessof a bromoacetyl adduct with a protein having free thiol groups asdescribed in U.S. Pat. No. 6,455,288. Free sulfhydryls may be providedin the form of free cysteine residues or by reducing protein disulfidebonds by a reagent such as dithiothreitol. Alternatively, thiol groupscan be added to a protein having free primary amino groups by reactingwith 2-iminothiolane (IT) in aqueous buffer, followed by removal ofunreacted IT. A detailed protocol for the thiolation of the protein KLHis provided in U.S. Pat. No. 5,439,798.

FIGS. 7A and 7B are schematic representation of preparation of reactiveforms of KLH and an enzyme according to an embodiment. FIG. 7Arepresents an exemplary bromoacetylation of KLH and an enzyme. FIG. 8Arepresents an exemplary thiolation of KLH and G6PDH. FIGS. 8A-K areschematic representations of synthetic schemes of exemplary gabapentinconjugates—enzyme (e.g., G6PDH) conjugates and KLH conjugates using KLHor enzyme (e.g., G6PDH) prepared in FIGS. 7A and 7B.

Anti-Gabapentin Antibodies

As noted above, the term “antibody” as used in the context of thepresent disclosure, refers to a specific binding partner of an analyte(e.g., gabapentin), and is meant to encompass whole antibodies as wellas antigen-binding fragments thereof (such as, for example, F(ab′)2,Fab′, Fab and Fv), naturally occurring antibodies, hybrid antibodies,chimeric antibodies, single-chain antibodies, and antibody fragmentsthat retain antigen binding specificity, and the like. Antibodies can beof any class (e.g., IgM, IgG, IgA, IgE; frequently IgG) and generatedfrom any source (although usually non-human, usually a non-human mammalsuch as a rabbit, mouse, rat, goat, etc.). Thus, “antibody” is meant toencompass not only intact immunoglobulin molecules, but also suchfragments and derivatives of immunoglobulin molecules as may be preparedby techniques known in the art, and retaining the antibody activity ofan intact immunoglobulin.

Antibodies may be derived from polyclonal compositions monoclonalcompositions. As noted above, “antibodies” is also meant to encompasssingle chain antibodies or scFvs, where such recombinantly producedantibody fragments retain the binding characteristics of the aboveantibodies. Recombinantly produced antibody fragments within the meaningof “antibody” generally include at least the VH and VL domains of thesubject antibodies, so as to retain the binding characteristics of thesubject antibodies. These recombinantly produced antibody fragments maybe readily prepared using any convenient methodology, such as themethodology disclosed in U.S. Pat. Nos. 5,851,829 and 5,965,371; thedisclosures of which are herein incorporated by reference.

Anti-gabapentin antibodies include those that bind one or moregabapentin epitopes. Anti-gabapentin antibodies thus include antibodiesthat bind, particularly that specifically bind, one or more of an amineepitope of a gabapentin moiety, a carboxylic acid epitope of agabapentin moiety, a cyclohexane epitope of a gabapentin moiety, or anycombination thereof (e.g., both an amine epitope and a carboxylic acidepitope, both an amine epitope and a cyclohexane epitope, both acarboxylic acid epitope and a cyclohexane epitope, or all threeepitopes). Anti-gabapentin antibodies may bind one or more ofunconjugated gabapentin, a gabapentin derivative, a gabapentinconjugate, or any combination thereof.

Producing Anti-Gabapentin Antibodies

Anti-gabapentin antibodies can be prepared by using an immunogenicgabapentin conjugate described herein and applying methods for antibodyproduction that are well known in the art. For examples of generaltechniques used in raising, purifying and modifying antibodies, and thedesign and execution of immunoassays, the reader is referred to Handbookof Experimental Immunology (D. M. Weir & C. C. Blackwell, eds.); CurrentProtocols in Immunology (J. E. Coligan et al., eds., 1991); David Wild,ed., The Immunoassay Handbook (Stockton Press N.Y., 1994); and R.Masseyeff, W. H. Albert, and N. A. Staines, eds., Methods ofImmunological Analysis (Weinheim: VCH Verlags gesellschaft mbH, 1993).

Antibodies obtained using any of the disclosed techniques are screenedor purified not only for their ability to react with gabapentin, but fora low cross-reactivity with potential interfering substances.“Cross-reactivity” may be determined in a quantitative immunoassay byestablishing a standard curve using known dilutions of the targetanalyte, gabapentin. The standard curve is then used to calculate theapparent concentration of the interfering substance present in variousknown amounts in samples assayed under similar condition. Thecross-reactivity is the apparent concentration divided by the actualconcentration multiplied by 100. An exemplary immunoassay fordetermining cross-reactivity is a homogeneous enzyme immunoassay using awild type G6PDH as described in U.S. Pat. No. 3,817,837 or mutant G6PDHengineered to contain a cysteine per subunit as described in U.S. Pat.Nos. 6,033,890, 6,090,567 and 6,455,288. Furthermore, thecross-reactivity can be determined in the same type of immunoassay inwhich the antibody will ultimately be used.

Producing Polyclonal Antibodies

Polyclonal antibodies that bind gabapentin may be raised byadministration of an immunogenic gabapentin conjugate to an animal host,usually mixed with an adjuvant. Any animal host which producesantibodies can be used. The immunogen is conveniently prepared forinjection by rehydrating lyophilized immunogen to form a solution orsuspension. Exemplary adjuvants are water-in-oil immersions,particularly Freund's complete adjuvant for the first administration,and Freund's incomplete adjuvant for booster doses. The preparation istypically administered in a variety of sites, and typically in two ormore doses over a course of at least 4 weeks. Serum is harvested andtested for the presence of anti-gabapentin antibody using agabapentin-protein conjugate or other gabapentin conjugates in astandard immunoassay or precipitation reaction.

Polyclonal antisera typically contain antibodies not reactive withgabapentin and cross-reactive with other substances. Methods forpurifying specific antibodies from a polyclonal antiserum are known inthe art. A particularly effective method is affinity purification usinga column of gabapentin conjugated to a solid phase. One manner ofpreparing a gabapentin column is to conjugate gabapentin or a gabapentinderivative to a protein other than the protein used in the immunogen,and then attach the conjugate to a commercially available activatedresin, such as CNBr-activated SEPHAROSE™. The anti-gabapentin antibodyis passed over the column, the column is washed, and the antibody iseluted with a mild denaturing buffer such as 0.1 M glycine, 0.2 M NaCl,pH 2.5.

Producing Monoclonal Antibodies

Anti-gabapentin monoclonal antibodies are prepared by a number ofdifferent techniques known in the art. For example, for hybridomatechnology, the reader is referred generally to Harrow & Lane (1988),U.S. Pat. Nos. 4,491,632, 4,472,500, and 4,444,887, and Methods inEnzymology, 73B:3 (1981). One common way to produce monoclonalantibodies is to immortalize and clone a splenocyte or otherantibody-producing cell recovered from an animal that has been immunizedagainst gabapentin as described earlier. The clone is immortalized by aprocedure such as fusion with a non-producing myeloma, by transfectingwith Epstein Barr Virus, or transforming with oncogenic DNA. The treatedcells are cloned and cultured, and clones are selected that produceantibody of the desired specificity. Specificity testing may beperformed on culture supernatants by a number of techniques, such asusing the immunizing antigen as the detecting reagent in an immunoassay.A supply of monoclonal antibody from the selected clone can then bepurified from a large volume of culture supernatant, or from the ascitesfluid of suitably prepared host animals injected with the clone. Theantibody may be tested for activity as raw supernatant or ascites, andis optionally purified using standard biochemical preparation techniquessuch as ammonium sulfate precipitation, ion exchange chromatography, andgel filtration chromatography.

Producing Fragments and Other Derivatives of Immunoglobulins

Fragments and other derivatives of immunoglobulins can be prepared bymethods of standard protein chemistry, for example, subjecting theantibody to cleavage with a proteolytic enzyme such as pepsin, papain,or trypsin; and reducing disulfide bonds with such reagents asdithiothreitol. Genetically, engineered variants of intactimmunoglobulin can be produced by obtaining a polynucleotide encodingthe antibody, and applying the general methods of molecular biology tosplice encoding sequences or introduce mutations and translate thevariant. Antibodies that are engineered variants of particular interestinclude chimeric and humanized antibodies, Fab-like fragments,single-chain variable region fragments (scFv), and diabodies.

Detectably Labeled Anti-Gabapentin Antibodies

The anti-gabapentin antibodies may also be labeled in order tofacilitate detection. A variety of protein labeling schemes are known inthe art and may be employed, the particular scheme and label chosenbeing the one most convenient for the intended use of the antibody, e.g.immunoassay.

Examples of labels include labels that permit both the direct andindirect measurement of the presence of the antibody. Examples of labelsthat permit direct measurement of the antibody include radiolabels, suchas ³H or ¹²⁵I, fluorescers, dyes, beads, chemiluminescers, colloidalparticles, and the like. Examples of labels which permit indirectmeasurement of the presence of the antibody include enzymes where asubstrate may provide for a colored or fluorescent product. For example,the antibodies may be labeled with a covalently bound enzyme capable ofproviding a detectable product signal after addition of suitablesubstrate. Instead of covalently binding the enzyme to the antibody, theantibody may be modified to comprise a first member of specific bindingpair which specifically binds with a second member of the specificbinding pair that is conjugated to the enzyme, e.g. the antibody may becovalently bound to biotin and the enzyme conjugate to streptavidin.Examples of suitable enzymes for use in conjugates include horseradishperoxidase, alkaline phosphatase, malate dehydrogenase and the like.Where not commercially available, such antibody-enzyme conjugates arereadily produced by techniques known to those skilled in the art.

Immunoassays

The present disclosure provides immunoassay methods for assessing thepresence or absence of gabapentin in a sample of interest. Due tovarious factors, including the pronounced inter-individual variabilityin gabapentin pharmacokinetics, immunoassays to assess gabapentin statusare of interest. Since gabapentin is excreted renally unchanged and isnot metabolized or at least does not produce detectable metabolites.Therefore, there are no pharmacogenomic issues that affect gabapentinconcentration and it is not subject to significant pharmacokinetic druginteractions with other drugs.

Immunoassays of the present disclosure can be of a variety of formats.The immunoassays may be separation immunoassays (also known asheterogeneous immunoassays) or homogeneous immunoassays. Furthermore,the immunoassays may be qualitative or quantitative. Assays of thisdisclosure include both sandwich and competition assays. Theimmunoassays may embody assays that are neither sandwich nor competitionassays, as in certain assays involving immunoprecipitation.

In general, the immunoassays of the present disclosure for detecting thepresence of absence of gabapentin in a sample can be conducted byadding, to a reaction mixture, (i) a sample suspected of containinggabapentin and (ii) an anti-gabapentin antibody capable of forming of acomplex of gabapentin that may be present in the sample and theantibody; and detecting the presence or absence of the complex. Thepresence or absence of said complex is indicative of the presence orabsence of gabapentin in said sample. Moreover, the amount of complexformed can be assessed to determine the concentration of gabapentinpresent in the sample (e.g., to provide an assessment of serum or tissueconcentration of gabapentin in a subject from whom the sample wasobtained). The presence and/or amount of complex can be assesseddirectly (e.g., by detecting bound antibody in the complex) orindirectly (e.g., by assessing activity of an enzyme in a gabapentinenzyme conjugate, where when the gabapentin enzyme conjugate is notbound to antibody, a detectable signal is generated, indicating that theanti-gabapentin antibody in the reaction mixture has been bound bygabapentin from the sample).

In general, the immunoassays of the disclosure entail combining thesample with an anti-gabapentin antibody under conditions that permit theformation of a stable complex between the analyte to be tested and theantibody.

Assays may be performed in solution or may use a solid (insoluble)support (e.g. polystyrene, nitrocellulose, or beads), using any standardmethods (e.g., as described in Current Protocols in Immunology, Coliganet al., ed.; John Wiley & Sons, New York, 1992). Typical methods includeELISAs (enzyme-linked immunosorbent assays), IRMAs (immunoradiometricassays), and RIAs (radioimmunoassays).

Where the assay is performed in solution, the test samples (and,optionally a control sample) is incubated with an anti-gabapentinantibody for a time period sufficient to allow formation of analyte andaffinity reagent complexes, for example, between about 0.1 hrs up to 24hrs, or more. As previously noted, the anti-gabapentin antibody mayinclude a detectable label (e.g. radionuclide, fluorescer, or enzyme).The sample is then treated to separate the gabapentin-anti-gabapentinantibody complexes from excess, unreacted anti-gabapentin antibody (e.g.by addition of an anti-anti-gabapentin antibody (e.g.,anti-immunoglobulin antiserum) followed by centrifugation to precipitatethe complexes, or by binding to an affinity surface such as a second,unlabelled anti-gabapentin antibody fixed to a solid substrate such asSepharose® or a plastic well). Detection of anti-gabapentin antibodybound to a gabapentin may be achieved in a variety of ways well known inthe art. If necessary, a substrate for the detectable label may be addedto the sample.

Where the assay uses a solid support, the support can have ananti-gabapentin antibody (or gabapentin conjugate) bound to a supportsurface. Binding of the assay reagent facilitates the stable,wash-resistant binding of gabapentin which may be present in the sample(or anti-gabapentin antibody that is not bound to gabapentin from thesample, and is present in the reaction mixture, as in a competitivebinding assay) to the solid support via specific binding to theanti-gabapentin antibody. The insoluble supports may be any compositionsto which antibodies or suitable gabapentin conjugates can be bound,which is readily separated from soluble material, and which is otherwisecompatible with the overall method of detection of anti-gabapentinantibody a sample.

The surface of such supports may be solid or porous and of anyconvenient shape. Examples of suitable insoluble supports to which theanti-gabapentin antibody is bound include beads, e.g. magnetic beads,membranes and microtiter plates. These can be composed of glass, plastic(e.g. polystyrene), polysaccharides, nylon or nitrocellulose.

Assay reagents can include the anti-gabapentin antibodies as disclosedherein, as well as anti-anti-gabapentin antibodies, which may beoptionally detectably labeled. Methods for binding antibodies or otherproteins to solid supports are well known in the art. After binding ofan assay reagent to the support, the support may be treated with ablocking agent, which binds to the support in areas not occupied by theassay reagent. Suitable blocking agents include non-interfering proteinssuch as bovine serum albumin, casein, gelatin, and the like.Alternatively, several detergents at non-interfering concentrations,such as Tween, NP40, TX100, and the like may be used. Such blockingtreatment reduces nonspecific binding.

Qualitative and Quantitative Methods

Assays of this disclosure include both qualitative and quantitativeassays. Typical quantitative methods involve mixing an analyte with apre-determined amount of the reagent antibody, and correlating theamount of complex formed with the amount of analyte in the originalsample using a relationship determined using standard samples containingknown amounts of analyte in the range expected for the sample to betested. In a qualitative assay, sufficient complex above or below athreshold level established by samples known to contain or be free ofanalyte establish the assay result. Unless otherwise stated, the act of“measuring” or “determining” in this disclosure refers alternately toqualitative and quantitative determination.

Samples

Samples may be biological samples taken from subjects suspected of beingadministered gabapentin.

As used herein, a “biological sample” refers to a sample of tissue orfluid isolated from a subject, which in the context of the inventiongenerally refers to samples suspected of containing gabapentin, whichsamples, after optional processing, can be analyzed in an in vitroassay.

Exemplary samples of interest include, but are not necessarily limitedto, a “blood sample” (which as used herein is meant to include wholeblood, plasma, serum, and the like), fecal matter, urine, tears, sweatsaliva, milk, organs, biopsies, secretions of the intestinal andrespiratory tracts, vitreous humor, and fluids obtainable during autopsy(such as cerebrospinal fluid). It should be noted that a “blood-derivedsample” refers to a sample that is prepared from blood or a fractionthereof, e.g., plasma or serum. Respiratory secretions (e.g., samplesobtained from fluids or tissue of nasal passages, lung, and the like),“Human serum”, as used herein, refers to the aqueous portion of humanblood remaining after the fibrin and suspended material (such as cells)have been depleted.

Blood samples, such as serum samples, can be obtained by any suitablemethod. In one embodiment, a trough serum/plasma is used and theconcentration range is 12-20 mg. Sweat samples can be obtained using,for example, a PharmChek® sweat patch from Sudormed. The PharmChek®sweat patch includes a semi-occlusive dressing containing a medicalgrade cellulose blotter paper collection pad, covered by a thin layer ofpolyurethane and acrylate adhesives. At the end of the wear period, thepad is eluted with a suitable buffer, such as 2.5 mL of 0.2 M acetatebuffer with methanol at pH 5.0 (25:75) or with acetonitrile.Furthermore, the biological samples may also be tissue samples, whichare extracted into liquid medium for immunoassay. For example, hairsamples can be tested by extracting into a liquid medium. The samplesmay be diluted or modified to facilitate the assay.

The samples may be experimental samples generated by any chemical orbiological method. For example, the samples may be standards containingknown concentrations of gabapentin or other substances used for assaycalibration.

In some embodiments, the biological sample will be diluted in a suitablesolution prior to assaying. In general, a solution suitable for dilutinga biological sample will include a buffer, such as phosphate bufferedsaline (PBS), and may include additional items, such as for example, anon-specific blocking agent, such as bovine serum albumin (BSA), adetergent, such as Triton-X-100, and the like.

Where desired, appropriate control samples for the assay include blood,serum, or urine collected from human subjects who have not receivedgabapentin (i.e., a negative control), or samples which contain a known,predetermined amount of a gabapentin analyte (i.e., a positive control).Alternatively, test results can be compared to detectable signal levelsknown to be associated with the presence or absence of gabapentin and/orcorrelated with an amount of gabapentin, e.g., a serum level ofgabapentin.

The assays may optionally include use of a calibration standard.“Calibration standard”, as used herein, refers to an aqueous mediumcontaining gabapentin at a predetermined concentration. In an exemplaryembodiment, a series of these calibration standards are available at aseries of predetermined concentrations. In another exemplary embodiment,the calibration standard is stable at ambient temperature. In yetanother exemplary embodiment, the calibration standards are in asynthetic matrix. In yet another exemplary embodiment, the calibrationstandards are in a non-synthetic matrix such as human serum.

In many embodiments, a suitable initial source for the human sample is ablood sample. As such, the sample employed in the subject assays isgenerally a blood-derived sample. The blood derived sample may bederived form whole blood or a fraction thereof, e.g., serum, plasma,etc., where in some embodiments the sample is derived from blood allowedto clot and the serum separated and collected to be used to assay.

In embodiments in which the sample is a serum or serum derived sample,the sample is generally a fluid sample. Any convenient methodology forproducing a fluid serum sample may be employed. In many embodiments, themethod employs drawing venous blood by skin puncture (e.g., fingerstick, venipuncture) into a clotting or serum separator tube, allowingthe blood to clot, and centrifuging the serum away from the clottedblood. The serum is then collected and stored until assayed. Once thepatient derived sample is obtained, the sample is assayed to determinethe level of gabapentin analyte.

Immunoassay Reagents

Immunoassay reagents that find use alone or in combination in the assaysdescribed herein include anti-gabapentin antibodies, gabapentinconjugates, and gabapentin (e.g., as a control or in competitive bindingassays). Immunoassay reagents can be provided in a buffered aqueoussolution. Such solutions may include additional components such assurface active additives, organic solvents, defoamers, buffers,surfactants, and anti-microbial agents. Surface active additives areintroduced to maintain hydrophobic or low-solubility compounds insolution, and stabilize components in the solution. Examples includebulking agents such as betalactoglobulin (BLG) or polyethyleneglycol(PEG); defoamers and surfactants such as Tween-20, Plurafac A38, TritonX-100, Pluronic 25R2, rabbit serum albumin (RSA), bovine serum albumin(BSA), and carbohydrates. Examples of organic solvents can includemethanol and other alcohols. Various buffers may be used to maintain thepH of the solution during storage. Illustrative buffers include HEPES,borate, phosphate, carbonate, tris, barbital and the like.Anti-microbial agents also extend the storage life of the immunoassayreagent.

Anti-Gabapentin Antibodies

Immunoassays generally involve at least one anti-gabapentin antibody,which may be produced by the methods disclosed herein. In an embodiment,the assays involve using an antibody raised against a gabapentinderivative-protein conjugate, particularly a low cross-reactivity withnon-gabapentin molecules that may be present in a reaction mixture.Anti-gabapentin antibodies can be polyclonal or monoclonal, morecommonly monoclonal, antibodies, capable of specifically bindinggabapentin.

Depending upon the assay format, the anti-gabapentin antibody can beoptionally detectably labeled, may be used in conjunction with asecondary antibody (i.e., an antibody that specifically binds ananti-gabapentin antibody) that may be detectably labeled. Exemplarydetectable labels for antibodies are described infra.

Gabapentin Conjugates

Gabapentin conjugates variously find use as immunoassay reagentsdepending on the assay format. For example, gabapentin conjugate can actas based on competitive binding reagent in competitive binding assays,or can provide for a detectable signal when not bound by ananti-gabapentin antibody (e.g., where the gabapentin conjugate is agabapentin G6PDH conjugate). Exemplary gabapentin conjugates useful asimmunoassay reagents are described below.

Detectable Labels

A variety of detectably labels can be used in connection with thegabapentin conjugate assay reagents for use in the methods disclosedherein. Such detectable labels can be isotopic labels. In otherembodiments, the detectable labels are non-isotopic signal-generatingmoieties, such as fluorophores and enzymes. Exemplary detectable labelsare described below. It will be apparent that while the detectablelabels are described below in the context of their use in gabapentinconjugates, many can also be adapted for use with anti-gabapentinantibodies.

Fluorophores

“Fluorophore” as used herein refers to moiety that itself fluoresces,can be made to fluoresce, or can provide for quenching of fluorescenceof a flurophor of a FRET pair (e.g., as in a FRET pair). In principle,any fluorophore can be used in the assays of this invention. In general,the fluorophore is selected so as to be compatible for use in the assayformat desired, and selected so as to be relatively insensitive to theassay conditions, e.g., pH, polarity, temperature and ionic strength.

Exemplary fluorophores can be characterized as having the followingcharacteristics: a. A fluorescence lifetime of greater than about 15nsec; b. An excitation wavelength of greater than about 350 nm; c. AStokes shift (a shift to lower wave-length of the emission relative toabsorption) of greater than about 20 nm; d. For homogeneous assaysdescribed below, fluorescence lifetime should vary with binding status;and e. The absorptivity and quantum yield of the fluorophore should behigh. The longer lifetime is advantageous because it is easier tomeasure and more easily distinguishable from the Raleigh scattering(background). Excitation wavelengths greater than 350 nm reducebackground interference because most fluorescent substances responsiblefor background fluorescence in biological samples are excited below 350nm. A greater Stokes shift also allows for less background interference.

The fluorophores generally have a functional group available forconjugation either directly or indirectly to a gabapentin intermediateto generate a gabapentin conjugate having the attached fluorophore.

Fluorophores for use in heterogeneous assays can be relativelyinsensitive to binding status. In contrast, fluorophores for use inhomogeneous assay can be sensitive to binding status, i.e., thefluorescence lifetime must be alterable by binding so that bound andfree forms can be distinguished.

Examples of fluorophores useful in the invention are naphthalenederivatives (e.g. dansyl chloride), anthracene derivatives (e.g.N-hydroxysuccinimide ester of anthracene propionate), pyrene derivatives(e.g. N-hydroxysuccinimide ester of pyrene butyrate), fluoresceinderivatives (e.g. fluorescein isothiocyanate), rhodamine derivatives(e.g. rhodamine isothiocyanate), phycoerythin, and Texas Red.

Enzymes

In an exemplary embodiment, the signal-generating moiety is an enzyme.From the standpoint of operability, a very wide variety of enzymes canbe used. But, as a practical matter, some enzymes have characteristicswhich make them more readily adaptable to the methods disclosed herein.

The enzyme can be selected so as to be stable to provide for desirableshelf-life, e.g., stable when stored for a period of at least threemonths or at least six months at temperatures which are convenient tostore in the laboratory, normally −20° C. or above. The enzyme can beselected so as to have a satisfactory turnover rate at or near the pHoptimum for binding to the antibody, this is normally at about pH 6-10,usually 6.0 to 8.0. A product of the enzymatic reaction facilitated bythe enzyme can be either formed or destroyed as a result of the enzymereaction, and can provide a enzyme reaction product which absorbs lightin the ultraviolet region or the visible region, that is the range ofabout 250-750 nm., usually 300-600 nm. The enzyme may also have asubstrate (including cofactors) which has a molecular weight in excessof 300, or in excess of 500. The enzyme which is employed or otherenzymes, with like activity, will not be present in the sample to bemeasured, or can be easily removed or deactivated prior to the additionof the assay reagents. Also, the enzyme can be selected so as to avoidthe impact of any naturally occurring inhibitors for the enzyme that maybe present in samples to be assayed or as some other component of thereaction mixture.

Although enzymes of up to 600,000 molecular weight can be employed,usually relatively low molecular weight enzymes will be employed of from10,000 to 300,000 molecular weight, more usually from about 10,000 to150,000 molecular weight, and frequently from 10,000 to 100,000molecular weight. Where an enzyme has a plurality of subunits themolecular weight limitations refer to the enzyme and not to thesubunits.

It may be desirable to select an enzyme that is susceptible todetectable labeling. In this instance, the enzyme can be detectablelabeled using appropriate detectable labels exemplified herein.

Exemplary enzymes include, but are not limited to: alkaline phosphatase,horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase,lactate dehydrogenase, β-galactosidase, and urease. Also, a geneticallyengineered fragment of an enzyme may be used, such as the donor andacceptor fragment of β-galactosidase utilized in CEDIA immunoassays(see, e.g., Henderson D R et al. Clin Chem. 32(9):1637-1641 (1986));U.S. Pat. No. 4,708,929. These and other enzymes which can be used havebeen discussed in detail by Eva Engvall in Enzyme Immunoassay ELISA andEMIT in Methods in Enzymology, 70:419-439 (1980) and in U.S. Pat. No.4,857,453.

In an exemplary embodiment, the enzyme is glucose-6-phosphatedehydrogenase (G6PDH) and it is attached to a gabapentin derivative,thus forming a gabapentin-reactive partner conjugate. An anti-gabapentinantibody used in conjunction with such gabapentin conjugates can beselected so as to specifically bind the gabapentin epitope presented bythe gabapentin enzyme conjugate, and thus affect activity of thegabapentin enzyme conjugate.

For assays employing gabapentin-enzyme conjugates, as an exemplaryprotein conjugate, in which a gabapentin derivative is labeled with anenzyme, the gabapentin derivative can be attached to the enzyme by anysuitable method. In certain embodiments, the chemistry described hereinfor formation of immunogenic protein conjugates of gabapentinderivatives is also used to prepare the enzyme conjugate. In this way,the gabapentin moiety presented to the antibody can more mirror thegabapentin epitope to which the antibody specifically binds.

The selection procedure is exemplified using a gabapentin-reactivepartner conjugate comprising G6PDH as the reactive partner and agabapentin derivative as the hapten. The first step in selecting aantibody involves testing the magnitude of antibody inhibition of agabapentin-reactive partner conjugate. In this step, the goal is todetermine and select for those antibodies which significantly inhibitthe enzyme activity of G6PDH. Antibodies which perform well in the firsttest are then subjected to a second test. Here, the antibody is firstincubated with gabapentin. Next the gabapentin-reactive partnerconjugate is added. An exemplary antibody preferentially binds togabapentin instead of the gabapentin-reactive partner conjugate. Thereduction in binding to the gabapentin-reactive partner conjugate wouldbe visible as an increase G6PDH activity.

Detection

Via Fluorescence

When a fluorescently labeled analyte (i.e., gabapentin antigen orantibody) is employed, the fluorescence emitted is proportional (eitherdirectly or inversely) to the amount of analyte. The amount offluorescence is determined by the amplitude of the fluorescence decaycurve for the fluorescent species. This amplitude parameter is directlyproportional to the amount of fluorescent species and accordingly to theanalyte.

In general spectroscopic measurement of fluorescence is accomplished by:a. exciting the fluorophore with a pulse of light; b. detecting andstoring an image of the excitation pulse and an image of all thefluorescence (the fluorescent transient) induced by the excitationpulse; c. digitizing the image; d. calculating the true fluorescenttransient from the digitized data; e. determining the amplitude of thefluorescent transient as an indication of the amount of fluorescentspecies.

According to the method, substantially all of the fluorescence emittedby the fluorescent species reaching the detector as a function of timefrom the instant of excitation is measured. As a consequence, the signalbeing detected is a superimposition of several component signals (forexample, background and one analyte specific signal). As mentioned, theindividual contributions to the overall fluorescence reaching thedetector are distinguished based on the different fluorescence decayrates (lifetimes) of signal components. In order to quantitate themagnitude of each contribution, the detected signal data is processed toobtain the amplitude of each component. The amplitude of each componentsignal is proportional to the concentration of the fluorescent species.

Via Enzyme

Detection of the amount of product produced by the gabapentin-reactivepartner conjugate of the invention can be accomplished by severalmethods which are known to those of skill in the art. Among thesemethods are colorimetry, fluorescence, and spectrophotometry. Thesemethods of detection are discussed in “Analytical Biochemistry” by DavidHolme, Addison-Wesley, 1998, which is incorporated herein by reference.

Solid Supports

The gabapentin conjugates and/or the anti-gabapentin antibodies to beused as reagents in an assay can be insolubilized by attachment to asolid phase. This can be, for example, a wall of a vessel containing thereagent, to a particulate, or to a large molecular weight carrier thatcan be kept in suspension but is removable by physicochemical means,such as centrifugation or microfiltration. The attachment need not becovalent, but is at least of sufficient permanence to withstand anyseparation techniques (including washes) that are part of the assayprocedure. Exemplary particulate materials include agarose, polystyrene,cellulose, polyacrylamide, latex particles, magnetic particles, andfixed red cells. Examples of commercially available matrices includeSepharose® (Pharmacia), Poros® resins (Roche Molecular Biochemicals),Actigel Superflow™ resins (Sterogene Bioseparations Inc.), andDynabeads™ (Dynal Inc.). The choice is not critical, and will generallydepend on such features as stability, capacity, accessibility of thecoupled antibody, flow rate (or the ability to disperse the resin in thereaction mixture), and ease of separation.

Assay Formats

As noted above, immunoassays for detection of gabapentin can be of avariety of formats. In general, the immunoassays involve combining oneor more immunoassay reagents (e.g., at least a anti-gabapentin antibody)with a test sample (i.e., a sample suspected of containing gabapentin)in a reaction mixture. “Reaction mixture” generally refers to thecombination of a sample suspected of containing gabapentin and one ormore immunoassay reagents as exemplified in the present disclosure tofacilitate detection of the presence or absence of gabapentin in thesample, where the detection may be qualitative or quantitative. Thereaction mixture is usually an aqueous solution, although theimmunoassay reagent(s) may be in solution or immobilized on a support(e.g., a substrate such as a bead). The reaction mixture can includeother components compatible with the immunoassay, e.g., buffers, and thelike.

Immunoassays usually are classified in one of several ways. For example,immunoassays can be classified according to the mode of detection used,i.e., enzyme immunoassays, radio immunoassays, fluorescence polarizationimmunoassays, chemiluminescence immunoassays, turbidimetric assays, etc.Another grouping method is according to the assay procedure used, i.e.,competitive assay formats, sandwich-type assay formats as well as assaysbased on precipitation or agglutination principles. In the instantapplication, a further distinction is made depending on whether washingsteps are included in the procedure (so-called heterogeneous assays) orwhether reaction and detection are performed without a washing step(so-called homogeneous assays). Exemplary assays are described in moredetail below.

Homogeneous and Heterogeneous Immunoassays

Immunoassays may be described as heterogeneous or homogeneous.“Homogeneous immunoassay”, as used herein, refers to an assay methodwhere the complex is typically not separated from unreacted reactioncomponents, but instead the presence of the complex is detected by aproperty which at least one of the reactants acquires or loses as aresult of being incorporated into the complex. Homogeneous assays knownin the art include systems involving fluorochrome and fluorochromequenching pairs on different reagents; enzyme and enzyme inhibitor pairson different reagents; chromophore and chromophore modifier pairs ondifferent reagents; and latex agglutination assays.

An exemplary homogeneous assay is the quantitative homogeneous enzymeimmunoassay in which a gabapentin moiety is conjugated to an activeenzyme. The conjugation is arranged so that the binding of ananti-gabapentin antibody to the derivative affects enzymatic activity ina qualitative or quantitative fashion. If a sample containing gabapentinis premixed with the antibody, the antibody will complex with thegabapentin and be prevented from binding to the enzyme conjugate. Inthis way, the activity of the enzyme can be correlated with the amountof gabapentin present in the sample.

G6PDH is an exemplary enzyme useful in such assays. In one embodiment,the G6PDH is a variant of a naturally-occurring G6PDH in which one ormore lysine residues are deleted or substituted, or one or more cysteineresidues are introduced. For example, Leuconostoc mesenteroides G6PDHare dimeric enzymes that have the ability to catalyze the oxidation ofD-glucose-6-phosphate to D-glucono-delta-lactone-6-phosphate byutilizing either NAD⁺ or NADP⁺. This property of using NAD⁺differentiates these enzymes from human G6PDH, which utilizes only NADP⁺effectively, and allows L. mesenteroides-specific G6PDH activity to bemeasured in the presence of human G6PDH, as for example in humansamples. G6PDHs from L. mesenteroides are used in current EMIT™homogeneous immunoassays (Syva Company, Palo Alto, Calif., U.S.A.). Twoexemplary genera of bacteria from which to select G6PDH are Leuconostocand Zymomonas. Within these genera L. mesenteroides, L. citreum, L.lactis, L. dextranicum, and Z. mobilis are of most interest, L.mesenteroides, L. citreum, L. lactis are specific examples.

FIG. 5 shows an exemplary scheme of a homogeneous, competitiveimmunoassay for gabapentin using an anti-gabapentin antibody and agabapentin-G6PDH enzyme conjugate in a plasma sample. In the absence ofplasma drug (A), an anti-gabapentin antibody binds to thegabapentin(H)-enzyme(G6PDH) conjugate and inactivates the enzyme. In thepresence of plasma drug (B), gabapentin from plasma, if present,competes with the gabapentin-G6PDH conjugate for binding to theantibody, thus allowing some fraction of the gabapentin-G6PDH conjugateto become active and convert NAD⁺ to NADH. The active G6PDH produces anabsorbance signal change over time at 340 nm. It is critical for thedrug of interest to be exposed to the antibody before thegabapentin-G6PDH conjugate; otherwise the antibody would becomesaturated with gabapentin-G6PDH conjugate and would be unable to detectthe drug. Therefore, the assay involves incubation of the sample andantibody before addition of the gabapentin-G6PDH conjugate.

Another example of a homogeneous assay system is the cloned enzyme donorimmunoassay, described in more detail below.

In a separation-based or “heterogeneous” assay, the detecting of acomplex of an anti-gabapentin antibody and an analyte involves a processwherein the complex formed is physically separated from either unreactedanalyte, unreacted antibody, or both.

In a heterogeneous immunoassay, a complex of an anti-gabapentin antibodyand an analyte may be first formed in the fluid phase, and thensubsequently captured by a solid phase reagent or separated on the basisof an altered physical or chemical property, such as by gel filtrationor precipitation. Alternatively, one of the reagents may be attached toa solid phase before contacting with other reagents, and then thecomplex may be recovered by washing the solid phase free of unreactedreagents. Separation-based assays typically involve use of a labeledderivative or antibody to facilitate detection or quantitation of thecomplex. Suitable labels include radioisotopes such as ¹²⁵I, enzymessuch as peroxidase and β-galactosidase, and fluorescent labels such asfluorescein isothiocyanate. The separation step involves removinglabeled reagent present in complex form from unreacted labeled reagent.The amount of label in the complex can be measured directly or inferredfrom the amount left unreacted.

Sandwich and Competition Assays

Assays of this disclosure include both sandwich and competition assays.Sandwich assays typically involve forming a complex in which the analyteto be measured is sandwiched between one reagent, such as a firstantibody used ultimately for separation of the complex, and anotherreagent, such as a second antibody used as a marker for the separatedcomplex. Competition assays involve a system in which the analyte to bemeasured competes with an derivative of the analyte for binding toanother reagent, such as an antibody. An example of a competition assayusing EMIT® is described in U.S. Pat. No. 3,817,837.

In one embodiment, the immunoassay further comprises adding a gabapentinconjugate comprising a gabapentin moiety and a detectable label to thesample. The presence or absence of gabapentin in the sample can bedetected by detecting the datable label. The detectable label maycomprise an enzyme and the detecting is by assaying activity of theenzyme. In an embodiment, the enzyme is a dehydrogenase, moreparticularly, G6PDH.

Lateral Flow Chromatography

The compounds and methods of the invention also encompass the use ofthese materials in lateral flow chromatography technologies. The essenceof lateral flow chromatography involves a membrane strip which comprisesa detection device, such as a non-isotopic signal generating moiety, forgabapentin. A sample from a patient is then applied to the membranestrip. The sample interacts with the detection device, producing aresult. The results can signify several things, including the absence ofthe gabapentin in the sample, the presence of the gabapentin in thesample, and even the concentration of the gabapentin in the sample.

In one embodiment, the invention provides a method of qualitativelydetermining the presence or absence of a gabapentin in a sample, throughthe use of lateral flow chromatography. The basic design of thequalitative lateral flow device is as follows: 1) The sample pad iswhere the sample is applied. The sample pad is treated with chemicalssuch as buffers or salts, which, when redissolved, optimize thechemistry of the sample for reaction with the conjugate, test, andcontrol reagents. 2) Conjugate release pad is typically a polyester orglass fiber material that is treated with a conjugate reagent such as anantibody colloidal gold conjugate. A typical process for treating aconjugate pad is to use impregnation followed by drying. In use, theliquid sample added to the test will redissolve the conjugate so that itwill flow into the membrane. 3) The membrane substrate is usually madeof nitrocellulose or a similar material whereby antibody capturecomponents are immobilized. 4) A wicking pad is used in tests whereblood plasma must be separated from whole blood. An impregnation processis usually used to treat this pad with reagents intended to conditionthe sample and promote cell separation. 5) The absorbent pad acts as areservoir for collecting fluids that have flowed through the device. 6)The above layers and membrane system are laminated onto a plasticbacking with adhesive material which serves as a structural member.

In another embodiment, the invention provides a method of qualitativelydetermining the presence of a gabapentin in a sample, through the use oflateral flow chromatography. In this embodiment, the membrane stripcomprises a sample pad, which is a conjugate release pad (CRP) whichcomprises a antibody that is specific for the gabapentin. This antibodyis conjugated to a non-isotopic signal-generating moiety, such as acolloidal gold particle. Other detection moieties useful in a lateralflow chromatography environment include dyes, colored latex particles,fluorescently labeled latex particles, non-isotopic signal generatingmoieties, etc. The membrane strip further comprises a capture line, inwhich the gabapentin derivative antigen is immobilized on the strip. Insome embodiments, this immobilization is through covalent attachment tothe membrane strip, optionally through a linker. In other embodiments,the immobilization is through non-covalent attachment to the membranestrip. In still other embodiments, the immobile gabapentin derivative inthe capture line is attached to a reactive partner, such as animmunogenic carrier like BSA.

Sample from a patient is applied to the sample pad, where it can combinewith the antibody in the CRP, thus forming a solution. This solution isthen allowed to migrate chromatographically by capillary action acrossthe membrane. When the gabapentin is present in the sample, agabapentin-antibody complex is formed, which migrates across themembrane by capillary action. When the solution reaches the captureline, the gabapentin-antibody complex will compete with the immobilegabapentin for the limited binding sites of the antibody. When asufficient concentration of gabapentin is present in the sample, it willfill the limited antibody binding sites. This will prevent the formationof a colored antibody-immobile gabapentin complex in the capture line.Therefore, absence of color in the capture line indicates the presenceof gabapentin in the sample.

In the absence of gabapentin in the sample, a colored antibody-immobilegabapentin complex will form once the solution reaches the capture lineof the membrane strip. The formation of this complex in the capture lineis evidence of the absence of gabapentin therapeutic in the sample.

In another embodiment, the invention provides a method of quantitativelydetermining the amount of a gabapentin in a sample, through the use oflateral flow chromatography. This technology is further described inU.S. Pat. Nos. 4,391,904; 4,435,504; 4,959,324; 5,264,180; 5,340,539;and 5,416,000, among others, which are herein incorporated by reference.In one embodiment, the antibody is immobilized along the entire lengthof the membrane strip. In general, if the membrane strip is made frompaper, the antibody is covalently bound to the membrane strip. If themembrane strip is made from nitrocellulose, then the antibody can benon-covalently attached to the membrane strip through, for example,hydrophobic and electrostatic interactions.

The membrane strip comprises a CRP which comprises the gabapentinattached to a detector moiety. In an exemplary embodiment, the detectormoiety is an enzyme, such as horseradish peroxidase (HRP).

Sample from a patient is applied to the membrane strip, where it cancombine with the gabapentin/detector molecule in the CRP, thus forming asolution. This solution is then allowed to migrate chromatographicallyby capillary action across the membrane. When the gabapentin is presentin the sample, both the sample gabapentin and the gabapentin/detectormolecule compete for the limited binding sites of the antibody. When asufficient concentration of gabapentin is present in the sample, it willfill the limited antibody binding sites. This will force thegabapentin/detector molecule to continue to migrate in the membranestrip. The shorter the distance of migration of the gabapentin/detectormolecule in the membrane strip, the lower the concentration ofgabapentin in the sample, and vice versa. When the gabapentin/detectormolecule comprises an enzyme, the length of migration of thegabapentin/detector molecule can be detected by applying an enzymesubstrate to the membrane strip. Detection of the product of the enzymereaction is then utilized to determine the concentration of thegabapentin in the sample. In another exemplary embodiment, the enzyme'scolor producing substrate such as a modified N,N-dimethylaniline isimmobilized to the membrane strip and 3-methyl-2-benzothiazolinonehydrazone is passively applied to the membrane, thus alleviating theneed for a separate reagent to visualize the color producing reaction.

Fluorescence Polarization Immunoassay for gabapentin

Fluorescence polarization immunoassay (FPIA) technology is based uponcompetitive binding between an antigen/drug in a sample and a knownconcentration of labeled antigen/drug. FPIA technology is described in,for example, U.S. Pat. Nos. 4,593,089, 4,492,762, 4,668,640, and4,751,190, which are incorporated herein by reference. Accordingly, theFPIA reagents, systems, and equipment described in the incorporatedreferences can be used with anti-gabapentin antibodies which are alsoanti-gabapentin analog antibodies.

The FPIA technology can be used to identify the presence of gabapentinand can be used in assays that quantify the amount of gabapentin in asample. In part, the rotational properties of molecules in solutionallow for the degree of polarization to be directly proportional to thesize of the molecule. Accordingly, polarization increases as molecularsize increases. That is, when linearly polarized light is used to excitea fluorescent-labeled or other luminescent-labeled gabapentin orderivative thereof, which is small and rotates rapidly in solution, theemitted light is significantly depolarized. When the fluorescent-labeledgabapentin or derivative interacts with or is bound to an antibody, therotation is slowed and the emitted light is highly polarized. This isbecause the antibody significantly and measurably increases the size ofthe complex. Also, increasing the amount of unlabeled gabapentin in thesample can result in decreased binding of the fluorescent-labeledgabapentin or derivative by the anti-gabapentin antibody, and therebydecrease the polarization of light emitted from sample. The quantitativerelationship between polarization and concentration of the unlabeledgabapentin in the sample can be established by measuring thepolarization values of calibrations with known concentrations ofgabapentin. Thus, FPIA can be used to identify the presence andconcentration of gabapentin in a sample.

In one embodiment, the assay involves an FPIA assay system. An exampleof components of the FPIA system can include the following: i)monoclonal or polyclonal anti-gabapentin antibodies capable ofspecifically binding to gabapentin and a gabapentin derivative; ii) asample suspected of containing the gabapentin; and iii) gabapentinderivative labeled with a fluorescent moiety, such as fluorescein.Alternatively, the system can be provided as a kit exclusive of thesample. Additionally, the system can include various buffercompositions, gabapentin concentration gradient compositions or a stockcomposition of gabapentin, and the like.

Homogeneous Microparticle Immunoassay for Gabapentin

Homogeneous microparticles immunoassay (“HMI”) technology, which can bereferred to as immunoturbidimetric assays, is based on the agglutinationof particles and compounds in solution. When particles and/or chemicalcompounds agglutinate, particle sizes can increase and increase theturbidity of a solution. Accordingly, anti-gabapentin antibodies can beused with microparticles and gabapentin derivatives in order to assessthe presence, and optionally the amount, of gabapentin in a sample. HMItechnologies can be advantageous because the immunoassays can beperformed on blood, blood hemolysate, serum, plasma, tissue, and/orother samples. HMI assays can be configured to be performed withgabapentin and/or a gabapentin derivative loaded onto a microparticle,or with an anti-gabapentin antibody loaded onto a microparticle. HMI orimmunoturbidimetric assays are well known in the art for measuringagglutination of substances in a sample.

Immunoturbidimetric assay technologies are described in, e.g., U.S. Pat.Nos. 5,571,728, 4,847,209, 6,514,770, and 6,248,597, which are includedherein by reference. Such assays involve light attenuation,nephelometric, or turbidimetric methods. The formation of anagglutinated compound AB from gabapentin (A) and anti-gabapentinantibody microparticle binding partner (B) can be measured by the changewhich occurs in the scattering or absorption of the incident lightdirected into the sample. Alternatively, the anti-gabapentin antibody(A) can bind with a gabapentin or derivative loaded microparticle. Whensuspendable particles having an immobilized binding partner are used,there is an enhancement of the effects, which makes it possible todetermine considerably lower gabapentin concentrations. Thesehomogeneous methods can be carried out quickly and simply, and permit,in particular, the automation of sample analyses as described in moredetail below.

Cloned Enzyme Donor Immunoassays for Gabapentin

Cloned enzyme donor Immunoassays (“CEDIA®”, Roche Diagnostics), as arebased upon the competition of gabapentin in the biological sample with agabapentin conjugate containing an inactive genetically engineeredenzyme-donor (“ED”) fragment such as from β-D-galactosidegalactohydrolase or β-galactosidase (“β-gal”) from E. coli, for bindingto an antibody capable of binding gabapentin. If gabapentin is presentin the sample it binds to the antibody, leaving the ED portion of theED-derivative conjugate free to restore enzyme activity ofβ-D-galactoside galactohydrolase or B gal in the reaction mixture so asto be capable of association with enzyme acceptor (“EA”) fragments. Theactive enzyme comprised of the ED and EA is then capable of producing aquantifiable reaction product when exposed to an appropriate substrate.A preferred substrate is chlorophenol red-β-D-galactopyranoside(“CPRG”), which can be cleaved by the active enzyme into galactose andCPR, wherein CPR is measured by absorbency at about wavelength 570 nm.In the instance gabapentin is not present in the sample, the antibodybinds to the ED-derivative conjugate, thereby inhibiting association ofthe ED fragments with the EA fragments and inhibiting restoration ofenzyme activity. The amount of reaction product and resultant absorbancechange are proportional to the amount of gabapentin in the sample.

Chemiluminescent Heterogeneous Immunoassays for Gabapentin

A competitive assay using chemiluminescent microparticle immunoassay(“CMIA”) technology can also be used to assess whether or not gabapentinis present in a sample. Various types of CMIA technologies are wellknown in the art of heterogeneous immunoassays for determining thepresence and/or amount of a chemical entity in a sample. CMIA assays caninclude the use of anti-gabapentin antibodies, which are capable ofbinding to gabapentin and its derivatives, which are coupled toparticles, such as magnetic particles or particles suitable forseparation by filtration, sedimentation, and/or other means.Additionally, a tracer, which can include a gabapentin derivative linkedto a suitable chemiluminescent moiety, can be used to compete with freegabapentin in the patient's sample for the limited amount ofanti-gabapentin antibody on the particle. After the sample, tracer, andantibody particles interact and a routine wash step has removed unboundtracer, the amount of tracer bound to antibody particles can be measuredby chemiluminescence, wherein chemiluminescence is expressed in RelativeLight Units (RULE). The amount of chemiluminescence is inversely relatedto the amount of free drug in the patient's sample and concentration isdetermined by constructing a standard curve using known values of thedrug.

Other Immunoassays for Gabapentin

The gabapentin derivatives, conjugates, antibodies, immunogens, and/orother conjugates described herein are also suitable for any of a numberof other heterogeneous immunoassays with a range of detection systemsincluding but not limited to enzymatic or fluorescent, and/orhomogeneous immunoassays including but not limited to rapid lateral flowassays, and antibody arrays, as well as formats yet to be developed.

While various immunodiagnostic assays have been described herein thatutilize the gabapentin derivatives, conjugates, antibodies, immunogensand/or tracers, such assays can also be modified as is well known in theart. As such, various modifications of steps or acts for performing suchimmunoassays can be made within the scope of the present invention.

Kits

The present disclosure also provides kits that find use in practicingthe subject methods, as described above. The kits of the presentinvention can comprise an anti-gabapentin antibody in a container, andmay comprise a gabapentin conjugate (e.g., for use in a competitivebinding assay, for use in an enzyme-based assay, and the like). The kitsmay also include a calibration standard and/or control standard usefulin performing the assay; and, optionally, instructions on the use of thekit. Kit components can be in a liquid reagent form, a lyophilized form,or attached to a solid support. The reagents may each be in separatecontainers, or various reagents can be combined in one or morecontainers depending on cross-reactivity and stability of the reagents.

The sample, suspected of containing a gabapentin, and a calibrationmaterial, containing a known concentration of the gabapentin, areassayed under similar conditions. Gabapentin concentration is thencalculated by comparing the results obtained for the unknown specimenwith results obtained for the standard. This is commonly done byconstructing a calibration or dose response curve.

Various ancillary materials may be employed in an assay in accordancewith the present invention. In an exemplary embodiment, buffers and/orstabilizers are present in the kit components. In another exemplaryembodiment, the kits comprise indicator solutions or indicator“dipsticks”, blotters, culture media, cuvettes, and the like. In yetanother exemplary embodiment, the kits comprise indicator cartridges(where a kit component is bound to a solid support) for use in anautomated detector. In still another exemplary embodiment, additionalproteins, such as albumin, or surfactants, particularly non-ionicsurfactants, may be included. In another exemplary embodiment, the kitscomprise an instruction manual that teaches a method of the inventionand/or describes the use of the components of the kit.

Reagents and buffers used in the assays can be packaged separately or incombination into kit form to facilitate distribution. The reagents areprovided in suitable containers, and typically provided in a packagealong with written instructions relating to assay procedures.

An embodiment of the present disclosure relates to a kit forconveniently determining the presence or the absence of gabapentin in asample. The kit may comprise an anti-gabapentin antibody and agabapentin calibration standard. The gabapentin calibration standard maycomprise calibration and control standards useful in performing theassay. The kits can also optionally comprise a conjugate comprising agabapentin moiety and a detectable signal. In an exemplary embodiment, adetectable signal of the conjugate is an enzyme. In yet anotherembodiment, the enzyme is glucose-6-phosphate dehydrogenase (G6PDH). Inone embodiment, the G6PDH is a variant of a naturally-occurring G6PDH inwhich one or more lysine residues are deleted or substituted, or one ormore cysteine residues are introduced.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Some of the examples have been performed viaexperiment and some are prophetic based on techniques, standards, andresults well known in the art. Also, it should be apparent that theinvention can include additional embodiments not illustrated by example.Additionally, many of the examples have been performed with experimentalprotocols well known in the art using the gabapentin derivatives,antigens, immunogens, and anti-gabapentin antibodies prepared inaccordance with the present invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g. amounts, temperature, etc.)but some experimental errors and deviations should be accounted for.Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Example 1 Preparation of 4-Amino Gabapentin Derivative (Formula III)

Compound 1 (13.8 gram, 0.1 mole) described in R. Funk, et al., JOC,(1983), 2632 and J. Stewart, et al., JACS, (1998), 354, which areincorporated herein by reference was converted to compound 2 asdescribed in the literature U.S. Pat. Publication No. 2007/0123591 andChinese Pat. CN1740161, which are incorporated herein by reference, inoverall yield of 40% as a white powder.

Compound 2 (4.22 grams, 20 mmole) was converted to compound 3 asdescribed in S. Raillard, et al., U.S. Pat. Publication No.2004/0014940, which is incorporated herein by reference, as a palepowder in 90% yield.

To a stirred solution of compound 3 (3.11 gram, 10 mmole) in methanol(100 ml) at −78° C. was bubbled ozone gas (1 ml/min) from an ozonegenerator for 1 hour. Nitrogen gas was then bubbled for 1 minute andmercaptomethanol (1 ml) was added to the reaction mixture and allowed tocome to room temperature by removing the cooling bath. The solvent wasthan removed under reduced pressure to give aldehyde 4 (2.95 grams,95%).

To a stirred saturated solution of anhydrous ammonia in methanol (10 ml)at 0° C. was added compound 4 (626 mg, 2 mmole). After 30 min., sodiumcyanoborohydride (60 mg) was added in portions of 20 mg over 1 hour.After the addition was completed the reaction was allowed to stir for anadditional 2 hours. To the reaction was added 1N HCl until the reactionwas acidic (pH 3-5). The reaction was then evaporated to dryness andextracted with THF, and evaporated to dryness to give compound 5 as asemi glass solid (450 mg, 71%).

To a stirred solution of the amine 5 (314 mg, 1 mmol) in THF (6 ml) wasadded bromo acetyl NHS ester (260 mg, 1.2 mmol). The mixture was stirredovernight and then diluted with water (10 ml). The mixture was thenextracted with DCM (3×30 ml). The combined DCM layers were washed withbrine (30 ml), dried over MgSO₄ and evaporated to dryness under vacuum.The crude product was then purified on a silica gel column (DCM:MeOH,95:5) to give the bromoacetyl 6 (410 mg, 94%) as a white foam. The foamwas dissolved in anhydrous DCM (10 ml) and HBr (1N in DCM, 0.5 ml) wasadded at 0° C. The reaction was stirred for 30 min and then evaporatedto dryness to give compound 6 HCl salt as a white powder (350 mg, 98%).

Compound 7 described in S. Danishefsky, et al., Tetrahedron (1998),12721 and J. Varghese, et al., WO 2006/010094 and WO 2005/087752, whichare incorporated herein by reference, (11.0 gram, 0.1 mol) was convertedto compound 8 as described in the literature U.S. Pat. Publication No.2007/0123591 and Chinese Pat. CN1740161, which are incorporated hereinby reference, in overall yield of 68% as an off white powder.

Compound 8 (8.44 grams, 40 mmol) was converted to compound 9 asdescribed in S. Raillard, et al., U.S. Pat. Publication No.2004/0014940, which is incorporated herein by reference, as a whitesolid in 85% yield.

To a stirred solution of compound 9 (4.75 gram, 20 mmol) in methanol(150 ml) at −78° C. was bubbled ozone gas (1 ml/min) from an ozonegenerator for 1 hour. Nitrogen gas was then bubbled for 1 minute andmercaptomethanol (1.5 ml) was added to the reaction mixture and allowedto come to room temperature by removing the cooling bath. The solventwas than removed under reduced pressure to give compound 10 (4.84 grams,84%).

To a stirred saturated solution of anhydrous ammonia in methanol (10 ml)at 0° C. was added compound 10 (855 mg, 3 mmol). After 30 min., sodiumcyanoborohydride (90 mg) was added in portions of 30 mg over 1 hour.After the addition was completed the reaction was allowed to stir for anadditional 2 hours. To the reaction was added 1N HCl until the reactionwas acidic (pH 3-5). The reaction was then evaporated to dryness andextracted with THF, and evaporated to dryness to give compound 11 as anoff white solid (429 mg, 50%).

To a stirred solution of the amine 11 (286 mg, 1 mmol) in anhydrous THF(8 ml) was added bromo acetyl NHS ester (260 mg, 1.2 mmol). The mixturewas stirred overnight and then diluted with water (15 ml). The mixturewas then extracted with DCM (3×30 ml). The combined DCM layers werewashed with brine (40 ml), dried (MgSO4) and evaporated to dryness undervacuum. The crude product was then purified on a silica gel column(DCM:MeOH, 90:10) to give the bromoacetyl 12 (345 mg, 85%) as a whitesolid. The solid was dissolved in anhydrous DCM (10 ml) and HBr (1N inDCM, 0.5 ml) was added at 0° C. The reaction was stirred for 30 min andthen evaporated to dryness to give compound 12 HCl salt as a whitepowder (325 mg, 95%).

To a cold stirred saturated solution of ethylenediamine (900 mg, 15mmol) in methanol (20 ml) at 0° C. was added compound 10 (855 mg, 3mmol). After 30 min., sodium cyanoborohydride (90 mg) was added inportions of 30 mg over 1 hour. After the addition was completed thereaction was allowed to stir overnight. To the reaction was added 1N HCluntil the reaction was acidic (pH 3-5). The reaction was then evaporatedto dryness and extracted with THF, and evaporated to dryness to givecompound 13 as white solid (395 mg, 40%).

The same reaction conditions were used as for the preparation ofcompound 12 to prepare compound 14 in 95% yield as a pale yellow solid.

The same reaction conditions were used as for the preparation ofcompound 13 to prepare compound 15 as a pale yellow solid in 35% yield.

Compound 15 (345 mg, 1 mmol) was dissolved in anhydrous DCM (10 ml) andHBr (1N in DCM, 0.5 ml) was added at 0° C. The reaction was stirred for30 min and then evaporated to dryness to give compound 16 HCl salt as apale yellow solid (233 mg, 95%.

The same reaction conditions were used as for the preparation ofcompound 15 to prepare compound 17 in 35% yield as a pale yellow solid.

The same reaction conditions were used as for the preparation ofcompound 16 to prepare compound 18 as a pale yellow solid in 95% yield.

Example 2 Preparation of Carboxyl Modified Gabapentin Derivative(Formula II)

To a stirred solution of compound 19 as described in Chinese Pat.CN1740161 and S. Raillard, et al., U.S. Pat. Publication No.2004/0014940, which are incorporated herein by reference, (2.71 gram, 10mmol) in THF (100 ml) was added DCC (2.27 gram, 11 mmol) and NHS (1.61gram, 14 mmol). The mixture was stirred for 6 hours. The diamine (7.60g, 50 mmol) and DIEA (4.0 ml, 22 mmol) were added at room temperatureand the reaction was stirred overnight. The solvent was then evaporatedto dryness under vacuum. To the residue was added water (60 ml) andextracted with ethyl acetate (2×50 ml). The combined organic layers werethen washed with HCl (1N, 20 ml), and saturated sodium bicarbonate (20ml) and dried (Na₂SO₄). The ethyl acetate was removed under reducedpressure to give the crude product. The crude product was purified on asilica gel column (EtOAc:hexane, 1:3) to give compound 20 (2.79 g, 85%)as a white solid.

To a stirred solution of compound 20 (329 mg, 1 mmol) in THF (5 ml) wasadded HCl (gas to saturate) at 0° C. The reaction was stirred for 1 hourand then was evaporated to dryness to give compound 21 as a pale yellowsolid (225 mg, 99%).

The same reaction conditions were used as for the preparation ofcompound 20 to prepare compound 23 in 80% yield as pale yellow foam.

The same reaction conditions were used as for the preparation ofcompound 21 to prepare compound 24 in 98% yield as a pale yellow solid.

Example 3 Preparation of Amine Modified Gabapentin Derivative (FormulaI)

To a stirred solution of gabapentin (1.71 gram, 10 mmol) in DMF (50 ml)and triethyl amine (2 ml) was added bromo acetyl NHS ester (2.60 gram,12 mmol). The mixture was stirred overnight and then diluted with water(100 ml). The mixture was acidified with 1N HCl and was then extractedwith ethyl acetate (3×60 ml). The combined ethyl acetate layers werewashed with brine (30 ml), dried (MgSO₄) and evaporated to dryness undervacuum. The crude product was then purified on a silica gel column(DCM:MeOH, 95:5) to give the bromoacetyl 25 (2.48 gram, 85%) as a whitefoam.

To a solution of compound 26 as described in D. Gopal, U.S. Pat.Publication No. 2005/0148792 and P. Rossi, WO 2002/074727, which areincorporated herein by reference, (2.26 grams, 10 mmol) andethylenediamine (3.0 grams, 50 mmol) in ethanol (100 ml) was addedactivated Pd (100 mg). The reaction mixture was then purged with N₂ gastwice and then the mixture container was filled with H₂ gas to 60 PSI.The mixture was stirred at room temperature overnight. The mixture wasthen passed through a pad of Celite and the pad was washed with ethanol(2×50 ml). The combined ethanol fractions were evaporated to dryness.The residue was purified on a silica gel column (DCM:MeOH, 90:10) togive compound 27 as a white powder (1.7 g, 60%).

To a stirred solution of the amine 27 (284 mg, 1 mmol) in anhydrous THF(8 ml) was added bromoacetyl NHS ester (260 mg, 1.2 mmol). The mixturewas stirred overnight and then diluted with water (20 ml). The mixturewas then extracted with DCM (3×30 ml). The combined DCM layers werewashed with brine (30 ml), dried (MgSO₄) and evaporated to dryness undervacuum. The residue was purified on a silica gel column (DCM:MeOH, 95:5)to give the t-butyl ester intermediate. The ester was dissolved in DCM(10 ml) and TFA (0.2 ml) was added at room temperature. The reaction wasstirred for 45 min and then evaporated to dryness to give compound 28 asthe TFA salt (279 mg, 80%).

The same reaction conditions were used as for the preparation ofcompound 27 to prepare compound 29 as a pale yellow solid (1.5 gram,50%).

A stirred solution of compound 29 (301 mg, 1 mmol) in DCM (20 ml) wastreated with TFA (0.2 ml) for 1 hour at room temperature. The solventwas then removed to give the TFA salt of compound 30 as a white solid.

The same reaction conditions were used as for the preparation ofcompounds 27 and 29 to prepare compound 31 in 50% yield as a pale yellowfoam.

The same reaction conditions were used as for the preparation ofcompound 30 to prepare compound 32 as the TFA salt.

Example 4 Conjugation: Preparation of Gabapentin-SH-KLH Immunogen (51)

a) Preparation of Thiolated KLH (35)

One vial of lyophilized KLH (Pierce, 21 mg) was reconstituted with 3 mLof phosphate buffer (0.1 M, 0.15 M NaCl, 1 mM EDTA, pH 8.0). The KLHsolution was transferred to a reaction vial. Immediately beforereaction, 6-8 mg of SATA (N-Succinimidyl-S-acetylthioacetate) wasdissolved in 0.5 mL of DMSO (results in ˜55 mM solution). 30 μl of theSATA solution was combined with 3.0 mL of protein solution (7 mg/mL).The contents were mixed and reaction incubated at room temperature forat least 30 minutes. A Sephadex G-50 column was equilibrated with twocolumn volumes of buffer (0.1 M phosphate, 0.15 M NaCl, pH 7.2-7.5). Thereaction mixture was applied to column. Fraction (500 μL) was collectedimmediately. The fractions that contain protein were identified bymeasuring absorbance at 280 nm. Protein fractions were pooled to give 12mL. Deacylation to generate a sulfhydryl for use in cross-linking wasaccomplished adding 1.2 mL deacetylation solution (0.5 M Hydroxylamine,25 mM EDTA in PBS, pH 7.2-7.5). Contents were mixed and reactionincubated for 2 hours at room temperature. Sephadex G-50 desaltingcolumn was used to purify the sulfhydryl-modified protein from thehydroxylamine in the deacetylation solution. The pooled fraction wasconcentrated to 2.6 mL (8 mg/mL) using Amicon concentrator. See FIG. 7B.

b) Conjugation with Thiolated KLH

Dithiothreitol (DTT, 1 mM) was added to thiolated KLH to ensurereduction of disulfide bonds. The solution was allowed to mix overnightat 4° C. 10.2 mg Bromoacetamido gabapentin hapten (25) was dissolved in0.2 mL DMF. Gabapentin hapten (25) DMF solution was added in 5 to 10 μLquantities to a solution of thiolated KLH (35). The reaction wascontinued overnight at 4° C. This solution was dialyzed against threechanges (2.0 liter each) of HEPES buffer (10 mM, pH 7.0, 1 mM EDTA).This procedure yielded immunogen (51). See FIG. 8H.

Gabapentin derivative (25) is used in this Example. This conjugationtechnique is generally applicable to all gabapentin haptens ContainingBromoacetamido—such as gabapentin derivatives (6), (12), (14), and (28).See FIGS. 8A, 8B, 8C, and 8I.

Example 5 Conjugation: Preparation of Gabapentin-SH-KLH Immunogen (43)

a) Preparation of Bromoacetyl KLH (33)

To a solution of KLH (20 mg) in NaH₂PO₄—Na₂HPO₄ buffer (pH=8.0, 0.1M,2.0 mL) at 4° C. (ice-bath) was added a solution of bromoacetic acid NHSester (5.8 mg, 0.024 mmol) in DMF (0.2 mL). The pH value was maintainedat 8.0. The reaction mixture was stirred in the cold-room (4° C.) for 16hours. The mixture was purified by a Sephadex G-50 column, eluting withNaH₂PO₄—Na₂HPO₄ buffer (pH=7.00, 0.025 M). The eluted fractions from thecolumn were monitored by UV at 280 nm. A clean separation betweenbromoacetyl-KLH and the hapten was obtained. Fractions containing theproduct were pooled together (8.0 mL) and concentrated to 3.0 mL ofbromoacetyl-KLH (33) by an Amicon concentrator for the next reaction.See FIG. 7A.

b) Preparation of Gabapentin KLH Immunogen (43)

2.0 mg hapten (16) was dissolved in 100 μL DMF. To a solution ofbromoacetyl-KLH (33) (3.0 mL, pH=7.00) was added the above hapten (16)solution slowly at 4 OC under nitrogen. The pH value was maintained at7.0. The reaction was stirred at 4° C. (cold room) for 16 hours. Thereaction mixture was separated using a Sephadex G-50 column equilibratedwith NaH₂PO₄—Na₂HPO₄ buffer (pH=7.0, 0.025 M). The UV detector at 280 nmmonitored the eluted fractions from the column. A clean separationbetween KLH immunogen and the hapten was obtained. Fractions containingprotein (43) were pooled to a total volume of 9 mL and concentrated to 5mL. The concentration of immunogen (43) was measured by using BCAProtein Concentration Assay. The Immunogen (43) had a concentration of3.5 mg/mL with a hapten number of 1280, and can be used for theimmunizations. See FIG. 8D.

Gabapentin SH derivative (16) is used in this Example. However, thisconjugation technique is generally applicable to gabapentin derivatives(18), (21), (24), (30), and (32). See FIGS. 8E, 8F, 8G, 8J and 8K.

Example 6 Conjugation: Native G6PDH and Gabapentin Containing ReactiveSulfhydryl Groups

Native G6PDH was buffer exchanged with 50 mM phosphate-1.0 mM EDTA, pH8.0. The protein solution was chilled in an ice bath and mixed withaliquots (30 μL) of a 0.2 M solution of N-hydroxysucinimidylbromoacetate in DMF. After incubation in ice bath for 1 hr enzymeactivity was measured. This addition of the DMF aliquots was continuedtill the enzyme activity was deactivated to 75±5% as compared to that ofthe native enzyme. The derivatized protein was found to contain 0.97moles of bromoacetamido groups per mole of the protein. The proteinsolution was mixed 40 fold molar excess of the thiol derivative of thehapten (16) in DMF (50 μL) and mixture stirred gently in a cold box for16 to 24 hours. Excess hapten (16) was separated from the enzyme-haptenconjugate by passing the reaction mixture over a column of Sephadex G 50in 50 mM phosphate, pH 7.0. The column fractions containing theenzyme-hapten conjugate were pooled by measuring absorption at 280 nm.

Gabapentin derivative (16) is used in this Example. However, thisconjugation technique is generally applicable to all gabapentin Haptensto all Haptens Containing Reactive Sulfhydryl Groups. Haptens (18),(21), (24), (30), and (32) can use this conjugation procedure.

Example 7 Conjugation: G6PDH Containing Reactive Sulfhydryl Groups

Conjugation G6PDH-SH was buffer exchanged with 50 mM phosphate-1.0 mMEDTA, pH 7.25. A solution of the protein (2 mL at 5 mg/mL) was thenmixed with a dithioerythreitol (25 mM final concentration in thephosphate-EDTA buffer) and mixture incubated at 4° C. for 16 hours. Theprotein solution was then buffer exchanged with 50 mM phosphate, 1.0 mMEDTA, 5 mM DTT, pH 7.25. The protein solution (2 mL at 5 mg/mL) wasmixed with 40 fold molar excess of a DMF solution (0.05 mL) of hapten(25) and reaction mixture stirred gently at 4° C. for 16 to 24 hours.Excess hapten (25) was separated from the enzyme-hapten conjugate bypassing the reaction mixture over a column of Sephadex G 50 in 50 mMphosphate, pH 7.0. The column fractions containing the enzyme-haptenconjugate were pooled by measuring absorption at 280 nm.

Gabapentin derivative (25) is used in this Example. However, thisconjugation technique is generally applicable to all gabapentin Haptensto all Haptens Containing Reactive Bromoacetamido-Groups. Haptens (6),(12), (14), (25), and (28), can use this conjugation procedure.

Example 8 Preparation of Gabapentin-SH-G6PDH (52)

Preparation of Thiolated G6PDH (36)

The N-succinimidyl S-acetylthioacetate (SATA) reagent was used tointroduce protected sulfhydryls into native G6PDH. 21 mg G6PDH wasdialyzed against phosphate buffer (0.1 M, 0.15 M NaCl, 1 mM EDTA, pH8.0). The dialyzed G6PDH was transferred to a reaction vial. Immediatelybefore reaction, 6-8 mg of SATA was dissolved in 0.5 mL of DMSO (resultsin ˜55 mM solution). Combined was 3.0 mL of protein solution (7 mg/mL)with 30 μl of the SATA solution. The contents were mixed and reactionincubated at room temperature for at least 60 minutes. A 15 mL SephadexG-50 column was equilibrated with two column volumes of buffer (0.1 Mphosphate, 0.15 M NaCl, pH 7.2-7.5). The reaction mixture was applied tocolumn. 1 mL fraction was collected. Fractions that contain protein wereidentified by measuring for those peaks having absorbance at 280 nm. 9mL sample volume was collected (approximating 21 mg enzyme). The enzymewas dialyzed against a bicarbonate buffer (100 mM, pH 9.0) to give freeSH groups.

The pH of the enzyme solution of (a) above was adjusted to 7.2 with 0.1MHCl. Gabapentin derivative (25) (11.8 mg) was dissolved in 200 μL DMF.Gabapentin (25) DMF solution was added in 5 to 10 μL quantities to asolution of 21 mg thiolated G6PDH. The reaction was continued overnightat 4° C. This solution was dialyzed against three changes (2.0 litereach) of HEPES buffer (10 mM, pH 7.0, 1 mM EDTA).

Gabapentin derivative (25) is used in this Example. However, thisconjugation technique is generally applicable to all gabapentin HaptensContaining Bromoacetamido-such as haptens (6), (12), (14), and (28) wereconjugated.

Example 9 Preparation of Polyclonal Antibodies Reactive to Gabapentin

Polyclonal sera from 6 live rabbit were prepared by injecting the animalwith immunogenic (51). This immunogenic formulation comprises 200 μg ofthe immunogen for the first immunization and 100 μg for all subsequentimmunizations. Regardless of immunogen amount, the formulation was thendiluted to 1 mL with sterile saline solution. This solution was thenmixed thoroughly with 1 mL of the appropriate adjuvant: Freund'sComplete Adjuvant for first immunization or Freund's Incomplete Adjuvantfor subsequent immunizations. The stable emulsion was subsequentlyinjected subcutaneously with a 19×1½ needle into New Zealand whiterabbits. Injections were made at 3-4 week intervals. Bleeds of theimmunized rabbits were taken from the central ear artery using a 19×1needle. Blood was left to clot at 37° C. overnight, at which point theserum was poured off and centrifuged. Finally, preservatives were addedin order to form the polyclonal antibody material. Rabbit polyclonalantibodies to gabapentin produced by the above procedure are designatedas #10925, #10926, #10927, #10928, #10929, and #10930. Rabbit polyclonalantibody #10930 is used in examples below.

The gabapentin antibodies and enzyme conjugates may be employed inassays for the detection of gabapentin. Either of the immunogens (37),(39), (41), (43), (45), (47), (49), (51), (53), (55) or (57) can beinjected into a mouse, sheep or rabbit to raise antibody.

Rabbit polyclonal antibody #10930 was screened for curve size,precision, and specificity. The obtained antibody was added into theantibody diluent to prepare the antibody reagent. The antibody reagentconsists of antibody as prepared above, buffer, stabilizers,preservatives, and the substrates for the enzyme conjugate NAD andglucose 6 phosphate. Enzyme conjugate comprising compound (38), (40),(42), (44), (46), (48), (50), (52), (54), (56) or (58) and G6PDH wasadded into the conjugate reagent to prepare the enzyme conjugatereagent. The enzyme conjugate reagent consists of the conjugate, buffer,stabilizers and preservatives. Enzyme conjugate (52) is used with rabbitpolyclonal antibody #10930 in examples below.

The gabapentin antibodies and enzyme conjugates may be advantageouslyused in a homogeneous assay format to detect gabapentin in samples. Ananalyzer (instrument) useful to set up the assay is Roche Cobas Mira(Roche Diagnostics). Gabapentin containing sample is incubated withantibody reagent followed by the addition of the enzyme conjugatereagent. The enzyme conjugate activity decreases upon binding to theantibody. The enzyme conjugate, which is not bound to the antibody,catalyzes the oxidation of glucose 6-phosphate (G6P). The oxidation ofG6P is coupled with the reduction of NAD⁺ to NADH, which can be measuredat 340 nm. The change in the absorbance at 340 nm can be measuredspectrophotometrically. The gabapentin concentration in a specimen canbe measured in terms of G6PDH activity. The increase in the rate at 340nm is due to the formation of NADH and is proportional to the enzymeconjugate activity. An assay curve is generated (FIG. 6) usinggabapentin spiked into negative calibrator matrix. The assay rateincreases with increasing the concentration of free drug in the sample.

This technique is generally applicable to produce polyclonal antibodiesto gabapentin derivatives.

Example 10 Assay Performance

Standard Curve

A series of known concentrations of gabapentin standards (ranging from 0to 10 μg/mL) were prepared gravimetrically in MES(2-(N-Morpholino)ethanesulfonic acid, 0.01 M, pH 5.5) formulated withEDTA, protein additive, detergent, antifoam agent, and preservative.Similarly, quality control samples were prepared (1.0 and 5.0 μg/mL).

Gabapentin was dissolved in methanol to give a stock solution of 1000μg/mL. Pooled human serum was aliquoted in 10 mL portions. Gabapentinstock solution was added to the aliquots in preparing a series of knownconcentrations of gabapentin calibrators ranging from 0 to 50 μg/mL.Antibody #10930 Reagent was prepared by adding antibody #10930 toantibody/substrate diluent. The antibody/substrate reagent was assayedwith Enzyme Conjugate Reagent (52). Calibration curves were generated onthe Cobas Mira® by assaying each level in duplicate. An example of thesecalibrator rates is shown in Table 1 and a typical plot is provided inFIG. 6.

TABLE 1 Calibrator Reaction Rate Gabapentin Reaction Rate (mA/min)Concentration (μg/mL) Average of Duplicates 0.00 519.2 5.00 602.3 10.00611.4 25.00 627.3 50.00 646.0 0.00 519.2Specificity of the Immunoassay

The specificity of the immunoassay was evaluated by adding potentiallycrossreactant drugs to human serum and determining the increase in theapparent concentration as a result of the presence of crossreactant.Separate stock solutions of gabapentin were prepared by dissolving thedrug in methanol to give a stock solution of 1000 μg/mL. 10 μg/mL ofcrossreactant plus 5 μg/mL of gabapentin was added to individual humanserum samples to give a final volume of 1 mL. Each sample was assayed induplicate. Testing was performed on the Cobas Mira Analyzer®. Thepercentage concentration above 5 μg/mL of gabapentin was calculated foreach crossreactant.

TABLE 2 Gabapentin Cross-Reactivity of Antibody with other Antiepileptic drugs Percent Increase in AED Apparent Gabapentin Gabapentin 0μg/mL (Control) 0% Gabapentin 5 μg/mL (Control) 98%  Lamotrigine 100μg/mL 0% Zonisamide 50 μg/mL 0% Levetiracetam 100 μg/mL 0% Carbamazepine100 μg/mL 0% Valproic Acid 100 μg/mL 0%

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure.

We claim:
 1. A compound having the structure:

wherein R₁, R₂, or R₃ is —X—W-L-Z, wherein when R₁ is —X—W-L-Z, X is NH,and R₂ is —OH, and R₃ is —H, when R₂ is —X—W-L-Z, X is NH, R₁ is —NH₂,and R₃ is —H, and when R₃ is —X—W-L-Z, X is a heteroatom or lower alkylgroup, R₁ is —NH₂, and R₂ is —OH, and W is a lower alkyl group or acarbonyl group; L is a linker or at least one bond between W and Z; andZ is an enzyme.
 2. The compound of claim 1, wherein said linkercomprises 0 to 40 carbon atoms and 0-6 heteroatoms.
 3. The compound ofclaim 2, wherein W is a lower alkyl and said linker is selected from thegroup consisting of —(CH₂)_(n)C(O)—, —C(O)(CH₂)_(n)—,—C(O)(CH₂)_(n)NH—C(O)—, —C(O)(CH₂)_(m)NH—C(O)(CH₂)_(n)—,—(CH₂)_(n)SCH₂C(O)—, —(CH₂)_(m)SCH₂C(O)(CH₂)_(n)—,—(CH₂)_(m)C(O)NH(CH₂)_(n)—, —(CH₂)_(n)NH—C(O)—,—(CH₂)_(m)NH—C(O)(CH₂)_(n)—, —C(O)—(CH₂)_(n)—, and —(CH₂)_(n)—; whereinm, n, o, and p are independently selected from an integer from 0 to 10.4. The compound of claim 2, wherein W is a carbonyl and said linker isselected from the group consisting of —(CH₂)_(n)C(O)—,—(CH₂)_(n)SCH₂C(O)—, —(CH₂)_(m)SCH₂C(O)(CH₂)_(n)—,—(CH₂)_(m)C(O)NH(CH₂)_(n)—, —(CH₂)_(n)NH—C(O)—,—(CH₂)_(m)NH—C(O)(CH₂)_(n)—, and —(CH₂)_(n)—; wherein m, n, o, and p areindependently selected from an integer from 0 to
 10. 5. The compound ofclaim 1, wherein W is a lower alkyl group and the lower alkyl group is amethyl group.
 6. The compound of claim 1, having the structure:

wherein Z, L, and W are as defined in claim
 1. 7. The compound of claim1, having the structure:

wherein W, L and Z are as defined in claim
 1. 8. The compound of claim1, having the structure:

wherein W, L and Z are as defined in claim
 1. 9. The compound of claim1, wherein said enzyme is a glucose-6-phosphate dehydrogenase (G6PDH).10. The compound of claim 9, wherein said G6PDH comprises at least onecysteine per subunit, wherein said cysteine is not native to anaturally-occurring G6PDH.
 11. The compound of claim 1, wherein saidenzyme is selected from alkaline phosphatase, β-galactosidase andhorseradish peroxidase.